From 9e9eea97337abfa8958d2af4f136b342831b338f Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Mon, 15 Jul 2013 13:55:44 -0400 Subject: [PATCH 01/14] mrnaseq foo --- doc/mrnaseq/de-novo-assembly.txt | 143 +++++++++++++++++++++++++++++++ doc/mrnaseq/index.txt | 10 +++ 2 files changed, 153 insertions(+) create mode 100644 doc/mrnaseq/de-novo-assembly.txt create mode 100644 doc/mrnaseq/index.txt diff --git a/doc/mrnaseq/de-novo-assembly.txt b/doc/mrnaseq/de-novo-assembly.txt new file mode 100644 index 0000000..ad3c901 --- /dev/null +++ b/doc/mrnaseq/de-novo-assembly.txt @@ -0,0 +1,143 @@ +=================================================== +Doing a small de novo mRNAseq assembly with Trinity +=================================================== + +Installing Trinity +------------------ + +To install Trinity:: + + cd /root + + curl -L http://sourceforge.net/projects/trinityrnaseq/files/latest/download?source=files > trinity.tar.gz + + tar xzf trinity.tar.gz + cd trinityrnaseq_r2013-02-25/ + export FORCE_UNSAFE_CONFIGURE=1 + make + +Install ctb Python packages +--------------------------- + +Install screed and khmer:: + + cd /usr/local/share + git clone https://github.com/ged-lab/screed.git + cd screed + python setup.py install + + cd /usr/local/share + git clone https://github.com/ged-lab/khmer.git + cd khmer + make + +Installing blastkit +------------------- + +Installing some prerequisites:: + + pip install pygr + apt-get -y install lighttpd + + cd /etc/lighttpd/conf-enabled + ln -fs ../conf-available/10-cgi.conf ./ + echo 'cgi.assign = ( ".cgi" => "" )' >> 10-cgi.conf + + /etc/init.d/lighttpd restart + +Next, install BLAST:: + + cd /root + + curl -O ftp://ftp.ncbi.nih.gov/blast/executables/release/2.2.24/blast-2.2.24-x64-linux.tar.gz + tar xzf blast-2.2.24-x64-linux.tar.gz + cp blast-2.2.24/bin/* /usr/local/bin + cp -r blast-2.2.24/data /usr/local/blast-data + +And put in blastkit:: + + cd /root + git clone https://github.com/ctb/blastkit.git -b ec2 + cd blastkit/www + ln -fs `pwd` /var/www/blastkit + + mkdir files + chmod a+rxwt files + chmod +x /root + +Install bowtie +-------------- + +Download and install bowtie:: + + cd /root + curl -O -L http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip + unzip bowtie-0.12.7-linux-x86_64.zip + cd bowtie-0.12.7 + cp bowtie bowtie-build bowtie-inspect /usr/local/bin + +Download and preparing the test data +------------------------------------ + +Grab the coral data:: + + cd /mnt + curl -O https://s3.amazonaws.com/public.ged.msu.edu/coral-settled-400k-pe.fq.gz + +Break the interleaved FASTQ data into left and right files for Trinity:: + + python /usr/local/share/khmer/sandbox/split-pe.py coral-settled-400k-pe.fq.gz + +Assemble! :: + + /root/trinityrnaseq_r2013-02-25/Trinity.pl --left coral-settled-400k-pe.fq.gz.1 --right coral-settled-400k-pe.fq.gz.2 --seqType fq -JM 5G + +Note that this last bit (5G) is the maximum amount of memory to use. You +can increase (or decrease) it based on what machine you rented. + +Are you impatient?? +~~~~~~~~~~~~~~~~~~~ + +Note: I have a little test database that you can install below INSTEAD of +doing the assembly:: + + mkdir /root/blastkit/db + curl https://s3.amazonaws.com/public.ged.msu.edu/coral-mini-assembly.fa.gz | gunzip > /root/blastkit/db/db.fa + +Set up a BLAST database +----------------------- + +Assuming everything is successful, let's make this BLASTable. + +Copy the assembly:: + + mkdir /root/blastkit/db + cp trinity_out_dir/Trinity.fasta /root/blastkit/db/db.fa + +Format the database for BLASTing:: + + cd /root/blastkit/db + formatdb -i db.fa -o T -p F + +Format the database for sequence retrieval:: + + python ../index-db.py db.fa + +Now, go to 'http:///blastkit/' and you should have a simple +BLAST interface. If you're using the coral dataset, above, try the following +query:: + + MSRADPGKNSEPSESKMSLELRPTAPSDLGRSNEAFQDEDLERQNTPGNSTVRNRVVQSGEQGHAKQDDRQITIEQEPLG + NKEDPEDDSEDEHQKGFLERKYDTICEFCRKHRVVLRSTIWAVLLTGFLALVIAACAINFHRALPLFVITLVTIFFVIWD + HLMAKYEQRIDDFLSPGRRLLDRHWFWLKWVVWSSLILAIILWLSLDTAKLGQQNLVSFGGLIMYLILLFLFSKHPTRVY + WRPVFWGIGLQFLLGLLILRTRPGFVAFDWMGRQVQTFLGYTDTGARFVFGEKYTDHFFAFKILPIVVFFSTVMSMLYYL + GLMQWIIRKVGWLMLVTMGSSPIESVVAAGNIFIGQTESPLLVQPYLPHVTKSELHTIMTAGFATIAGSVLGAYISFGVS + STHLLTASVMSAPAALAVAKLFWPETEKPKITLKSAMKMENGDSRNLLEAASQGASSSIPLVANIAANLIAFLALLSFVN + SALSWFGSMFNYPELSFELICSYIFMPFSFMMGVDWQDSFMVAKLIGYKTFFNEFVAYDHLSKLINLRKAAGPKFVNGVQ + QYMSIRSETIATYALCGFANFGSLGIVIGGLTSIAPSRKRDIASGAMRALIAGTIACFMTACIAGILSDTPVDINCHHVL + ENGRVLSNTTEVVSCCQNLFNSTVAKGPNDVVPGGNFSLYALKSCCNLLKPPTLNCNWIPNKL + +.. rename sequences +.. add default query name on blastkit +.. e-mail mccauley +.. password protect diff --git a/doc/mrnaseq/index.txt b/doc/mrnaseq/index.txt new file mode 100644 index 0000000..afd6086 --- /dev/null +++ b/doc/mrnaseq/index.txt @@ -0,0 +1,10 @@ +The Eel Pond mRNAseq Tutorial +============================= + +Contents: + +.. toctree:: + :maxdepth: 1 + + de-novo-assembly + From e62038b4014fcff8c129bb7a72dcf3f91def2b19 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Wed, 31 Jul 2013 17:02:23 -0400 Subject: [PATCH 02/14] basic mrnaseq foo --- doc/mrnaseq/0-download-and-save.txt | 78 +++++ doc/mrnaseq/1-quality.txt | 354 ++++++++++++++++++++++ doc/mrnaseq/2-diginorm.txt | 118 ++++++++ doc/mrnaseq/coral.txt | 7 + doc/mrnaseq/de-novo-assembly.txt | 13 +- doc/mrnaseq/images/AttachVolume.png | Bin 0 -> 51196 bytes doc/mrnaseq/images/AttachVolumeWindow.png | Bin 0 -> 45750 bytes doc/mrnaseq/images/ClickVolumes.png | Bin 0 -> 83673 bytes doc/mrnaseq/images/CreateVolume.png | Bin 0 -> 84340 bytes doc/mrnaseq/images/CreateVolumeWindow.png | Bin 0 -> 32117 bytes doc/mrnaseq/index.txt | 10 +- doc/mrnaseq/rsem.txt | 96 ++++++ doc/mrnaseq/saving-data-persistently.txt | 114 +++++++ doc/mrnaseq/using-screen.txt | 58 ++++ 14 files changed, 840 insertions(+), 8 deletions(-) create mode 100644 doc/mrnaseq/0-download-and-save.txt create mode 100644 doc/mrnaseq/1-quality.txt create mode 100644 doc/mrnaseq/2-diginorm.txt create mode 100644 doc/mrnaseq/coral.txt create mode 100644 doc/mrnaseq/images/AttachVolume.png create mode 100644 doc/mrnaseq/images/AttachVolumeWindow.png create mode 100644 doc/mrnaseq/images/ClickVolumes.png create mode 100644 doc/mrnaseq/images/CreateVolume.png create mode 100644 doc/mrnaseq/images/CreateVolumeWindow.png create mode 100644 doc/mrnaseq/rsem.txt create mode 100644 doc/mrnaseq/saving-data-persistently.txt create mode 100644 doc/mrnaseq/using-screen.txt diff --git a/doc/mrnaseq/0-download-and-save.txt b/doc/mrnaseq/0-download-and-save.txt new file mode 100644 index 0000000..222fb03 --- /dev/null +++ b/doc/mrnaseq/0-download-and-save.txt @@ -0,0 +1,78 @@ +=========================================== +0. Downloading and Saving Your Initial Data +=========================================== + +The basics +---------- + +Amazon is happy to rent disk space to you, in addition to compute time. +They'll rent you disk space in a few different ways, but the way that's +most useful for us is through what's called Elastic Block Store. This +is essentially a hard-disk rental service. + +There are two basic concepts -- "volume" and "snapshot". A "volume" can +be thought of as a pluggable-in hard drive: you create an empty volume of +a given size, attach it to a running instance, and voila! You have extra +hard disk space. Volume-based hard disks have two problems, however: +first, they cannot be used outside of the "availability zone" they've +been created in, which means that you need to be careful to put them +in the same zone that your instance is running in; and they can't be shared +amongst people. + +Snapshots, the second concept, is the solution to transporting and +sharing the data on volumes. A "snapshot" is essentially a frozen +copy of your volume; you can copy a volume into a snapshot, and a +snapshot into a volume. + +Getting started +--------------- + +Run through :doc:`saving-data-persistently` once, to get the hang of +the mechanics. Essentially you create a disk; attach it; format it; +and then copy things to and from it. + +Downloading and saving your data to a volume +-------------------------------------------- + +There are *many* different ways of getting big sequence files to and +from Amazon. The two that I mostly use are 'curl', which downloads +files from a Web site URL; and 'ncftp', which is a robust FTP client +that let's you get files from an FTP site. Sequencing centers almost +always make their data available in one of these two ways. + +.. note:: + + To use ncftp on your Amazon instance, you may need to install it:: + + apt-get -y install ncftp + +For example, to retrieve a file from an FTP site, you would do something +like:: + + cd /mnt + ncftp -u ftp://path/to/FTP/site + +use 'cd' to find the right directory, and then:: + + >> mget * + +to download the files. Then type 'quit'. You can also use 'curl' to +download files one at a time from Web or FTP sites. + +Once you have the files, figure out their size using 'du -sk' (e.g. after the +above, 'du -sk /mnt' will tell you how much data you have saved under /mnt), +and go create and attach a volume (see :doc:`saving-data-persistently`). + +This data is now something that will *stick around* when you shut down +your instance. It's a good rule of thumb to do "savepoints" -- whenever +you complete a big chunk of work, think about saving the data at that +point. I've broken the mRNAseq tutorial down into chunks of work where +you can do this -- after each Web page, basically. + +Some test data +-------------- + +To get started with multfile analysis and assembly, I've provided some +test data. It's on snapshot 'snap-f5a9dea7', so go create a volume from +that and mount it as '/data' to get started. + diff --git a/doc/mrnaseq/1-quality.txt b/doc/mrnaseq/1-quality.txt new file mode 100644 index 0000000..3b41984 --- /dev/null +++ b/doc/mrnaseq/1-quality.txt @@ -0,0 +1,354 @@ +================================================ +1. Quality Trimming and Filtering Your Sequences +================================================ + +.. note:: + + This follows the NGS 2013 tutorial, + :doc:`../tutorials-2013/short-read-quality-evaluation`, but for multiple + files. + +.. note:: + + The end results of this tutorial are available as public snapshot + snap-8b155fd9 on EC2/EBS. + +Install software +---------------- + +Install screed:: + + cd /usr/local/share + git clone https://github.com/ged-lab/screed.git + cd screed + python setup.py install + +Install the bleeding-edge version of khmer:: + + cd /usr/local/share + git clone https://github.com/ged-lab/khmer.git -b bleeding-edge + cd khmer + make + + echo 'export PYTHONPATH=/usr/local/share/khmer/python' >> ~/.bashrc + source ~/.bashrc + + +Install Trimmomatic:: + + cd /root + curl -O http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/Trimmomatic-0.27.zip + unzip Trimmomatic-0.27.zip + cp Trimmomatic-0.27/trimmomatic-0.27.jar /usr/local/bin + +Install libgtextutils and fastx:: + + cd /root + curl -O http://hannonlab.cshl.edu/fastx_toolkit/libgtextutils-0.6.1.tar.bz2 + tar xjf libgtextutils-0.6.1.tar.bz2 + cd libgtextutils-0.6.1/ + ./configure && make && make install + + cd /root + curl -O http://hannonlab.cshl.edu/fastx_toolkit/fastx_toolkit-0.0.13.2.tar.bz2 + tar xjf fastx_toolkit-0.0.13.2.tar.bz2 + cd fastx_toolkit-0.0.13.2/ + ./configure && make && make install + +In each of these cases, we're downloading the software -- you can use +google to figure out what each package is and does if we don't discuss +it below. We're then unpacking it, sometimes compiling it (which we +can discuss later), and then installing it for general use. + +Find your data +-------------- + +Either load in your own data (as in :doc:`0-download-and-save`) or +create a volumne from snapshot snap-f5a9dea7 and mount it as /data. +Check:: + + ls /data + +If you see all the files you think you should, good! Otherwise, debug. + +If you're using the Tulin et al. data provided in the snapshot above, +you should see:: + + /data/0Hour_ATCACG_L002_R1_001.fastq.gz + +Link your data into a working directory +--------------------------------------- + +Rather than *copying* the files into the working directory, let's just +*link* them in -- this creates a reference so that UNIX knows where to +find them but doesn't need to actually move them around. :: + + cd /mnt + mkdir work + cd work + + ln -fs /data/*.fastq.gz . + +(The 'ln' command is what does the linking.) + +Download the Illumina adapters +------------------------------ + +In the working directory, :: + + curl -O https://s3.amazonaws.com/public.ged.msu.edu/illuminaClipping.fa + +.. note:: + + You'll need to make sure these are the right adapters for your data. + If they are, you should see that some of them are trimmed off, below; + if they're not, you shouldn't see anything get trimmed. + +Adapter trim each pair of files +------------------------------- + +(From this point on, you may want to be running things inside of +screen, so that you detach and log out while it's running; see +:doc:`using-screen` for more information.) + +If you're following along using the Nematostella data, you should have a +bunch of files that look like this (use 'ls' to show them):: + + 24HourB_GCCAAT_L002_R1_001.fastq.gz + ^^ + +Each file with an R1 in its name should have a matching file with an R2 -- +these are the paired ends. + +.. note:: + + You'll need to replace and , below, with the + names of your actual R1 and R2 files. You'll also need to replace + with something that's unique to each pair of files. + It doesn't really matter what, but you need to make sure it's different + for each pair of files. + +For *each* of these pairs, run the following:: + + # make a temp directory + mkdir trim + cd trim + + # run trimmomatic + java -jar /usr/local/bin/trimmomatic-0.27.jar PE s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 + + # interleave the remaining paired-end files + python /usr/local/share/khmer/sandbox/interleave.py s1_pe s2_pe | gzip -9c > ../.pe.fq.gz + + # combine the single-ended files + cat s1_se s2_se | gzip -9c > ../.se.fq.gz + + # go back up to the working directory and remove the temp directory + cd .. + rm -r trim + + # make it hard to delete the files you just created + chmod u-w *.pe.fq.gz *.se.fq.gz + +You can read the '#' comments above, but, briefly, this set of commands: + + * creates a temporary directory, 'trim/' + + * runs 'Trimmomatic' in that directory to trim off the adapters, and then + puts remaining pairs (most of them!) in s1_pe and s2_pe, and any orphaned + singletons in s1_se and s2_se. + + * interleaves the paired ends and puts them back in the working directory + + * combines the orphaned reads and puts them back in the working directory + +Automating things a bit +~~~~~~~~~~~~~~~~~~~~~~~ + +OK, once you've done this once or twice, it gets kind of tedious, doesn't it? +I've written a script to write these commands out automatically. Run it +like so:: + + cd /mnt/work + python /usr/local/share/khmer/sandbox/write-trimmomatic.py > trim.sh + +Run this, and then look at 'trim.sh' using the 'more' command -- :: + + more trim.sh + +If it looks like it contains the right commands, you can run it by doing:: + + bash trim.sh + +.. note:: + + This is a prime example of scripting to make your life much easier + and less error prone. Take a look at this file sometime -- + 'more /usr/local/share/khmer/sandbox/write-trimmomatic.py' to get + some idea of how this works. + +Quality trim each pair of files +------------------------------- + +After you run this, you should have a bunch of '*.pe.fq.gz' files and +a bunch of '*.se.fq.gz' files. The former are files that contain paired, +interleaved sequences; the latter contain single-ended, non-interleaved +sequences. + +Next, for each of these files, run:: + + gunzip -c | fastq_quality_filter -Q33 -q 30 -p 50 | gzip -9c > .qc.fq.gz + +This uncompresses each file, removes poor-quality sequences, and then +recompresses it. Note that (following :doc:`../tutorials-2013/short-read-quality-evaluation`) you can also trim things by putting in a 'fastx_trimmer -Q33 -l 70 |' into the mix. + +Automating this step +~~~~~~~~~~~~~~~~~~~~ + +This step can be automated with a 'for' loop at the shell prompt. Try:: + + for i in *.pe.fq.gz *.se.fq.gz + do + echo working with $i + newfile=`basename $i .fq.gz` + gunzip -c $i | fastq_quality_filter -Q33 -q 30 -p 50 | gzip -9c > ${newfile}.qc.fq.gz + done + +What this loop does is: + + - for every file ending in pe.fq.gz and se.fq.gz, + + - print out a message with the filename, + + - construct a name 'newfile' that omits the trailing .fq.gz + + - uncompresses the original file, passes it through fastq, recompresses it, + and saves it as "newfile".qc.fq.gz + +Extracting paired ends from the interleaved files +------------------------------------------------- + +The fastx utilities that we're using to do quality trimming aren't +paired-end aware; they're removing individual sequences. Because the +pe files are interleaved, this means that there may now be some orphaned +sequences in there. Downstream, we will want to pay special attention +to the remaining paired sequences, so we want to separate out the pe +and se files. How do we go about that? Another script, of course! + +The khmer script 'strip-and-split-for-assembly.py' does exactly that. +You run it on an interleaved file that may have some orphans, and it +produces .pe and .se files afterwards, containing pairs and orphans +respectively. + +To run it on all of the pe qc files, do:: + + for i in *.pe*.qc.fq.gz + do + python /usr/local/share/khmer/sandbox/strip-and-split-for-assembly.py $i + done + +Finishing up +------------ + +You should now have a whole mess of files. For example, in the Nematostella +data, for *each* of the original input files, you'll have:: + + 24HourB_GCCAAT_L002_R1_001.fastq.gz - the original data + 24HourB_GCCAAT_L002_R2_001.fastq.gz + 24HourB_GCCAAT_L002_R1_001.pe.fq.gz - adapter trimmed pe + 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz - FASTX filtered + 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.pe - FASTX filtered PE + 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.se - FASTX filtered SE + 24HourB_GCCAAT_L002_R1_001.se.fq.gz - adapter trimmed orphans + 24HourB_GCCAAT_L002_R1_001.se.qc.fq.gz - FASTX filtered orphans + +Yikes! What to do? + +Well, first, you can get rid of the original data. You already have it on a +disk somewhere, right? :: + + rm *.fastq.gz + +Next, you can get rid of the 'pe.fq.gz' and 'se.fq.gz' files, since you +only want the QC files. So:: + + rm *.pe.fq.gz *.se.fq.gz + +And, finally, you can toss the pe.fq.gz files, because you've turned *those* +into .pe and .se files. + + rm *.pe.qc.fq.gz + +So now you should be left with only three files for each sample:: + + 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.pe - FASTX filtered PE + 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.se - FASTX filtered SE + 24HourB_GCCAAT_L002_R1_001.se.qc.fq.gz - FASTX filtered orphans + +Things to think about +~~~~~~~~~~~~~~~~~~~~~ + +Note that the filenames, while ugly, are conveniently structured with the +history of what you've done. This is a good idea. + +Also note that we've conveniently named the files so that we can remove +the unwanted ones en masse. This is a good idea, too. + +Renaming files +-------------- + +I'm a fan of keeping the files named somewhat sensibly, and keeping them +compressed. Let's do some mass renaming:: + + for i in *.pe.qc.fq.gz.pe + do + newfile=`basename $i .pe.qc.fq.gz.pe`.pe.qc.fq + mv $i $newfile + gzip $newfile + done + +and also some mass combining:: + + for i in *.pe.qc.fq.gz.se + do + otherfile=`basename $i .pe.qc.fq.gz.se`.se.qc.fq.gz + gunzip -c $otherfile > combine + cat $i >> combine + gzip -c combine > $otherfile + rm $i + done + +and finally:: + + chmod u-w *.qc.fq.gz + +to make sure you don't accidentally delete something. + +Saving the files +---------------- + +At this point, you should save these files, which will be used in two +ways: first, for assembly; and second, for mapping, to do quantitation +and ultimately comparative expression analysis. You can save them by +doing this:: + + mkdir save + mv *.qc.fq.gz save + du -sk save + +This puts the data you want to save into a subdirectory named 'save', and +calculates the size. + +Now, create a volume of the given size -- divide by a thousand to get +gigabytes, multiply by 1.1 to make sure you have enough room, and then +follow the instructions in :doc:`saving-data-persistently`. Once +you've mounted it properly (I would suggest mounting it on /save +instead of /data!), then do :: + + rsync -av save /save + +which will copy all of the files over from the ./save directory onto the +'/save' disk. Then 'umount /save' and voila, you've got a copy of the files! + +Next stop: :doc:`2-digital-normalization`. + diff --git a/doc/mrnaseq/2-diginorm.txt b/doc/mrnaseq/2-diginorm.txt new file mode 100644 index 0000000..af330ed --- /dev/null +++ b/doc/mrnaseq/2-diginorm.txt @@ -0,0 +1,118 @@ +================================= +2. Applying Digital Normalization +================================= + +.. note:: + + You can start this tutorial with the contents of EC2/EBS snapshot + snap-126cc847. + +.. note:: + + You'll need ~15 GB of RAM for this, or more if you have a LOT of data. + +Link in your data +----------------- + +Make sure your data is in /mnt/work/. If you've loaded it onto /data, +you can do:: + + cd /mnt + mkdir work + cd /mnt/work + ln -fs /data/*.qc.fq.gz . + +Run digital normalization +------------------------- + +Apply digital normalization to the paired-end reads:: + + python /usr/local/share/khmer/scripts/normalize-by-median.py -p -k 20 -C 20 -N 4 -x 3e9 --savehash normC20k20.kh *.pe.qc.fq.gz + +and then to the single-end reads:: + + python /usr/local/share/khmer/scripts/normalize-by-median.py -k 20 -C 20 -N 4 -x 3e9 --loadhash normC20k20.kh --savehash normC20k20.kh *.se.qc.fq.gz + +Note the '-p' in the first normalize-by-median command -- when run on +PE data, that ensures that no paired ends are orphaned. However, it +will complain on single-ended data, so you have to give the data to it +separately. + +Also note the '-N' and '-x' parameters. These specify how much memory +diginorm should use. The product of these should be less than the +memory size of the machine you selected. The maximum needed for *any* +transcriptome should be in the ~60 GB range, e.g. -N 4 -x 15e9; for +only a few hundred million reads, 16 GB should be plenty. (See +`choosing hash sizes for khmer +`__ +for more information.) + +Trim off likely erroneous k-mers +------------------------------- + +Now, run through all the reads and trim off low-abundance parts of +high-coverage reads:: + + /usr/local/share/khmer/scripts/filter-abund.py -V normC20k20.kh *.keep + +This will turn some reads into orphans, but that's ok -- their partner +read was bad. + +Rename files +~~~~~~~~~~~~ + +You'll have a bunch of 'keep.abundfilt' files -- let's make things prettier. + +First, let's break out the orphaned and still-paired reads:: + + for i in *.pe.*.abundfilt; + do + python /usr/local/share/khmer/sandbox/strip-and-split-for-assembly.py $i + done + +We can combine the orphaned reads into a single file:: + + for i in *.se.qc.fq.gz.keep.abundfilt + do + pe_orphans=`basename $i .se.qc.fq.gz.keep.abundfilt`.pe.qc.fq.gz.keep.abundfilt.se + newfile=`basename $i .se.qc.fq.gz.keep.abundfilt`.se.qc.keep.abundfilt.fq.gz + cat $i $pe_orphans | gzip -c > $newfile + done + +We can also rename the remaining PE reads & compress those files:: + + for i in *.abundfilt.pe + do + newfile=`basename $i .fq.gz.keep.abundfilt.pe`.keep.abundfilt.fq + mv $i $newfile + gzip $newfile + done + +This leaves you with a whole passle o' files, most of which you want to go +away! + + 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz + 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz.keep + 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz.keep.abundfilt + 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz.keep.abundfilt.se + 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz + 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz.keep + 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz.keep.abundfilt + +And, finally, let's get rid of a lot of the old files :: + + rm *.se.qc.fq.gz.keep.abundfilt + rm *.pe.qc.fq.gz.keep.abundfilt.se + rm *.keep + rm *.abundfilt + rm *.qc.fq.gz + +Gut check +~~~~~~~~~ + +You should now have:: + + 6Hour_CGATGT_L002_R1_005.pe.qc.keep.abundfilt.fq.gz + 6Hour_CGATGT_L002_R1_005.se.qc.keep.abundfilt.fq.gz + +Save all these files to a new volume, and get ready to assemble! diff --git a/doc/mrnaseq/coral.txt b/doc/mrnaseq/coral.txt new file mode 100644 index 0000000..673378e --- /dev/null +++ b/doc/mrnaseq/coral.txt @@ -0,0 +1,7 @@ + +java -jar /usr/local/bin/trimmomatic-0.27.jar PE P_astreoides_larva_GCCAAT_L005_R?_001.fastq.gz s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 + + +mkdir larva +root@ip-10-40-10-213:/mnt/two# mv s?_?e larva/ +root@ip-10-40-10-213:/mnt/two# java -jar /usr/local/bin/trimmomatic-0.27.jar PE P_astreoides_settled*_L005_R?_001.fastq.gz s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 diff --git a/doc/mrnaseq/de-novo-assembly.txt b/doc/mrnaseq/de-novo-assembly.txt index ad3c901..31e3382 100644 --- a/doc/mrnaseq/de-novo-assembly.txt +++ b/doc/mrnaseq/de-novo-assembly.txt @@ -30,6 +30,9 @@ Install screed and khmer:: git clone https://github.com/ged-lab/khmer.git cd khmer make + + echo export PYTHONPATH=/usr/local/share/khmer/python >> ~/.bashrc + source ~/.bashrc Installing blastkit ------------------- @@ -39,9 +42,12 @@ Installing some prerequisites:: pip install pygr apt-get -y install lighttpd +and configure them:: + cd /etc/lighttpd/conf-enabled ln -fs ../conf-available/10-cgi.conf ./ echo 'cgi.assign = ( ".cgi" => "" )' >> 10-cgi.conf + echo 'index-file.names += ( "index.cgi" ) ' >> 10-cgi.conf /etc/init.d/lighttpd restart @@ -59,7 +65,7 @@ And put in blastkit:: cd /root git clone https://github.com/ctb/blastkit.git -b ec2 cd blastkit/www - ln -fs `pwd` /var/www/blastkit + ln -fs $PWD /var/www/blastkit mkdir files chmod a+rxwt files @@ -136,8 +142,3 @@ query:: SALSWFGSMFNYPELSFELICSYIFMPFSFMMGVDWQDSFMVAKLIGYKTFFNEFVAYDHLSKLINLRKAAGPKFVNGVQ QYMSIRSETIATYALCGFANFGSLGIVIGGLTSIAPSRKRDIASGAMRALIAGTIACFMTACIAGILSDTPVDINCHHVL ENGRVLSNTTEVVSCCQNLFNSTVAKGPNDVVPGGNFSLYALKSCCNLLKPPTLNCNWIPNKL - -.. rename sequences -.. add default query name on blastkit -.. e-mail mccauley -.. password protect diff --git 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zI;}z)y>)_4&I21(*VxfbbDAkq%rThyj6P3RW#V|E^UwR)4aNJ=k4gd!s0!&*l$2Hi z3|Z8#$Tz~BtDM3KyCox#T|sS*1}fLBOWXU{@;fZi2xk_1gG@_k z=v*4kIHX(+gfNNzZT7Dq=Kv>@PBQ3R#E5gd)>ULiaeCDw>Si n%Q5c<7E{qJnOSHr-@ifd(lMp>nN`n&f&Qe#`__ +to do some expression analysis. To install it:: + + cd /root + + curl -O http://deweylab.biostat.wisc.edu/rsem/src/rsem-1.2.5.tar.gz + + tar xzvf rsem-1.2.5.tar.gz + cd rsem-1.2.5 + make + +And now add this directory into your PATH, which is where Unix looks for +things to run:: + + echo 'export PATH=$PATH:/root/rsem-1.2.5' >> /root/.bashrc + source /root/.bashrc + +Installing bowtie +----------------- + +If you didn't install bowtie on this machine already (e.g. as part of +:doc:`de-novo-assembly`), RSEM needs it; do:: + + cd /root + curl -O -L http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip + unzip bowtie-0.12.7-linux-x86_64.zip + cd bowtie-0.12.7 + cp bowtie bowtie-build bowtie-inspect /usr/local/bin + +Running rsem +------------ + +Go to a working directory on /mnt:: + + cd /mnt + mkdir rsem + cd rsem + +Now, grab the reference transcriptome:: + + curl https://s3.amazonaws.com/public.ged.msu.edu/coral-mini-assembly.fa.gz | gunzip > coral.fa + + +and some reads:: + + curl -O http://athyra.idyll.org/~t/larva-ct-800k.fq.gz + curl -O http://athyra.idyll.org/~t/settled-ct-800k.fq.gz + +You'll need to uncompress the reads:: + + gunzip *.gz + +Now, prepare the reference for RSEM:: + + rsem-prepare-reference coral.fa coral + +Here, 'coral.fa' can be one of several files (comma-separated) in case +you have multiple different reference data sets. In this case it's +the transcriptome you generated, but there are lots of options (see +`the docs for rsem-prepare-reference +`__). + +And, finally, calculate the expression levels for the 'settled' and 'larva' +data sets:: + + rsem-calculate-expression settled-ct-800k.fq coral settled + rsem-calculate-expression larva-ct-800k.fq coral larva + +(See `the rsem-calculate-expression docs `__ for more info.) + +This outputs a couple files; the two most interesting, for now, are +'larva.genes.results' and 'settled.genes.results'. These contain the +following data:: + + head larva.genes.results + +should show you :: + + gene_id transcript_id(s) length effective_length expected_count TPM FPKM + comp0_c0_seq1 comp0_c0_seq1 291.00 291.00 3.00 102.12 164.47 + ... + +If you've installed Dropbox (see +:doc:`../tutorials-2013/installing-dropbox`), you can now copy the +files to Dropbox:: + + cp larva.genes.results settled.genes.results ~/Dropbox/ + +and you can open them directly in Excel as tab-delimited files. diff --git a/doc/mrnaseq/saving-data-persistently.txt b/doc/mrnaseq/saving-data-persistently.txt new file mode 100644 index 0000000..6fc8ab5 --- /dev/null +++ b/doc/mrnaseq/saving-data-persistently.txt @@ -0,0 +1,114 @@ +===================================== +Storing data persistently with Amazon +===================================== + +:Author: Rosangela Canino-Koning +:Date: June 7, 2010 + +If you want to save your data across instances -- that is, if you want +to have persistent data -- Amazon can do that for you, too. You need +to use the Amazon Elastic Block Storage service, which creates a virtual +hard drive that you can (virtually) attach to your EC2 instance. + +Note that EBS charges you based partly on how much disk space you have +allocated per month, rather than on connectivity or anything else, so even if +you don't have an EC2 machine running, you'll be paying Amazon for data +storage. + +You can read more about EBS `here `__. + +Prerequisites +------------- + +This tutorial assumes you've already set up an account on Amazon Web +Services, and that you've completed the EC2 tutorial to set up an +Amazon instance. + +Ask Amazon to create a new Elastic Block Storage Volume for you +--------------------------------------------------------------- + +At the AWS Management Console, on the left menu bar, click "Volumes". + +.. image:: images/ClickVolumes.png + :width: 90% + + +Click "Create Volume". + +.. image:: images/CreateVolume.png + :width: 90% + +Enter the desired size, and select the zone in which your instance is +running. The volume and instance must be in the same zone. Otherwise, +the volume cannot be attached to your instance. + +Then click "Create". + +.. image:: images/CreateVolumeWindow.png + :width: 90% + + +Wait for your volume to finish being created, then click "Attach Volume". + +.. image:: images/AttachVolume.png + :width: 90% + +Select the desired running instance. It will ask you for a device name +to attach; this should be '/dev/sdf', '/dev/sdg', etc. You can name it +anything up to at least 'i' or 'j'. Remember this for later -- it's +how the computer will know which disk to store data on! + +Click "Attach". + +.. image:: images/AttachVolumeWindow.png + :width: 90% + +When attachment is complete, connect to your instances via SSH. + +If the volume is newly created, you must format the volume. +**WARNING: ONLY DO THIS ONCE, WHEN YOU FIRST CREATE THE VOLUME. OTHERWISE, YOU WILL LOSE ALL YOUR +DATA**. +:: + + mkfs -t ext2 /dev/xvdf + +(If you used 'sdg' above, make it 'xvdg' etc. I know it's confusing.) + +It will ask you if you want to use the entire device -- say "y" for "yes. + +Then, mount the volume. You'll do this every time you attach the volume to an instance:: + + mkdir /data + mount /dev/xvdf /data + +Your drive is now ready to use -- it will be available under /data. +Files copied into that directory or directories underneath it will +be stored on your EBS volume. + +Shutting down your instance +--------------------------- + +Any volumes you have attached will automatically detach when you shut +down the instance. You can also stop all processes that are using the +volume, change out of the directory, and type :: + + cd + umount /data + +and then detach the volume via the AWS Web site. + +Snapshotting your volume +------------------------ + +Snapshots are backups of your volume that you can share with other people. +Snapshots are much more reliable long-term than volumes are, and you can use +them as a basis for creating a new volume (in which case the new volume will +start out containing all the data in the snapshot). So, if you upload some +raw data and want to work with it over a few weeks, we suggest: + + - create a volume and load the data onto the volume + - snapshot the original volume + - make a new volume from the snapshot, and delete the original volume + +You'll only be charged money for storing the snapshot and any *differences* +from the snapshot on the volumes based on that snapshot. diff --git a/doc/mrnaseq/using-screen.txt b/doc/mrnaseq/using-screen.txt new file mode 100644 index 0000000..aa45c06 --- /dev/null +++ b/doc/mrnaseq/using-screen.txt @@ -0,0 +1,58 @@ +============== +Using 'screen' +============== + +:Author: Rosangela Canino-Koning +:Date: June 9, 2011 +:Last Updated: July 24, 2013 + +Persistent Sessions +------------------- + +Screen is a window manager for terminal sessions. Screen allows you to +run a terminal session, and then disconnect from the computer, and be +able to return to the session at a later date. + +To start screen, you run the screen command with a few options:: + + screen -S + +Where *sessionname* is any meaningful or descriptive title for your screen +session. This creates an independent terminal session, and connects you to it. + +Most commands within screen are composed of a prefix key-stroke, +followed by a command character. By default, the prefix is Ctrl-A. In +this tutorial Ctrl-A will represented by "C-a". + +Let's try a few screen commands. + +To disconnect from the session (while leaving it running!), type:: + + C-a d + +This session will remain active until you choose to end it, or you +reboot the computer. You can at this point safely disconnect from SSH, +and the screen session will continue to run. + +To reconnect to the session, make sure you're logged into the UNIX machine, +and type:: + + screen -r + +To illustrate managing multiple screen session, disconnect from the current +session, and create a new session with a second name.:: + + C-a d + screen -S + +Disconnect from the second session, and then list the available sessions:: + + C-a d + screen -list + +Note, typing *screen -r* with multiple active screen sessions will display +the same information. + +To reconnect to the first session, include its name after the -r.:: + + screen -r From ed3eb112374a4b8257dc8bc75b8e816cbcb3e427 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Thu, 1 Aug 2013 10:39:45 -0400 Subject: [PATCH 03/14] put license terms everywhere --- README.txt | 21 ++++++++++++++++++++- doc/_templates/layout.html | 11 ++++++++++- 2 files changed, 30 insertions(+), 2 deletions(-) diff --git a/README.txt b/README.txt index 9d43711..ea80cf2 100644 --- a/README.txt +++ b/README.txt @@ -1,6 +1,25 @@ Documentation for the MSU Next-Gen Sequence Analysis Course, and other things, too. +As per http://ged.msu.edu/angus/, + +License: + +This documentation and all textual/graphic site content is licensed +under the Creative Commons - 0 License (CC0). Please feel free to +copy, modify, distribute and perform the work, even for commercial +purposes, all without asking permission. + +You can find the source code for this material under git version +control on github at https://github.com/ngs-docs/edda. Please fork at +your own leisure :) + +However, presentations (PPT/PDF) and PDFs are the property of their +respective owners and are under the terms indicated within the +presentation. If no terms are indicated, please do not reuse without +specific and explicit permission - i.e. all presentations and PDFs are +Copyright (C) their authors, all rights reserved, unless otherwise +stated. + Titus Brown ctb@msu.edu - diff --git a/doc/_templates/layout.html b/doc/_templates/layout.html index be088a5..4854fd8 100644 --- a/doc/_templates/layout.html +++ b/doc/_templates/layout.html @@ -43,6 +43,16 @@ {%- endif %}

{% block body %} {% endblock %} +
+LICENSE: +This documentation and all textual/graphic site content is licensed +under the +Creative Commons - 0 License +(CC0) -- fork @ +github. Presentations (PPT/PDF) and PDFs are the property of +their respective owners and are under the terms indicated within the +presentation. +
{{ comments() }}
@@ -50,7 +60,6 @@ {%- endif %} - {%- endblock %}
From 645190ed1c2dac9f93a6fb35ee3c75674a4c210f Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Wed, 7 Aug 2013 11:07:33 -0400 Subject: [PATCH 04/14] updated with partitioning --- doc/mrnaseq/0-download-and-save.txt | 22 ++++- doc/mrnaseq/1-quality.txt | 20 ++-- doc/mrnaseq/2-diginorm.txt | 12 ++- doc/mrnaseq/3-big-assembly.txt | 75 ++++++++++++++ .../5-building-transcript-families.txt | 97 +++++++++++++++++++ doc/mrnaseq/index.txt | 4 + 6 files changed, 213 insertions(+), 17 deletions(-) create mode 100644 doc/mrnaseq/3-big-assembly.txt create mode 100644 doc/mrnaseq/5-building-transcript-families.txt diff --git a/doc/mrnaseq/0-download-and-save.txt b/doc/mrnaseq/0-download-and-save.txt index 222fb03..1a407bf 100644 --- a/doc/mrnaseq/0-download-and-save.txt +++ b/doc/mrnaseq/0-download-and-save.txt @@ -2,10 +2,19 @@ 0. Downloading and Saving Your Initial Data =========================================== +We're going to run transcriptome assembly completely in the cloud, +because that way (a) you don't need to buy a big computer, and (b) +I don't have to figure out all the special details of your own +computer system. + +This does mean that the first thing you need to do is get your data +over to the cloud. I tend to just store it there in the first place, +because... + The basics ---------- -Amazon is happy to rent disk space to you, in addition to compute time. +...Amazon is happy to rent disk space to you, in addition to compute time. They'll rent you disk space in a few different ways, but the way that's most useful for us is through what's called Elastic Block Store. This is essentially a hard-disk rental service. @@ -19,7 +28,7 @@ been created in, which means that you need to be careful to put them in the same zone that your instance is running in; and they can't be shared amongst people. -Snapshots, the second concept, is the solution to transporting and +Snapshots, the second concept, are the solution to transporting and sharing the data on volumes. A "snapshot" is essentially a frozen copy of your volume; you can copy a volume into a snapshot, and a snapshot into a volume. @@ -73,6 +82,9 @@ Some test data -------------- To get started with multfile analysis and assembly, I've provided some -test data. It's on snapshot 'snap-f5a9dea7', so go create a volume from -that and mount it as '/data' to get started. - +test mRNAseq data from embryonic stages of *Nematostella vectensis*; +the source is `this excellent paper +`__ by Tulin et al., "A +quantitative reference transcriptome for Nematostella vectensis". The +data is on snapshot 'snap-f5a9dea7', so go create a volume from that +and mount it as '/data' to get started. diff --git a/doc/mrnaseq/1-quality.txt b/doc/mrnaseq/1-quality.txt index 3b41984..9b52b96 100644 --- a/doc/mrnaseq/1-quality.txt +++ b/doc/mrnaseq/1-quality.txt @@ -2,6 +2,10 @@ 1. Quality Trimming and Filtering Your Sequences ================================================ +Boot up an m1.xlarge machine from Amazon Web Services; this has about +15 GB of RAM, and 2 CPUs, and will be enough to complete the assembly +of the Nematostella data set. + .. note:: This follows the NGS 2013 tutorial, @@ -33,7 +37,6 @@ Install the bleeding-edge version of khmer:: echo 'export PYTHONPATH=/usr/local/share/khmer/python' >> ~/.bashrc source ~/.bashrc - Install Trimmomatic:: cd /root @@ -64,7 +67,10 @@ Find your data -------------- Either load in your own data (as in :doc:`0-download-and-save`) or -create a volumne from snapshot snap-f5a9dea7 and mount it as /data. +create a volume from snapshot snap-f5a9dea7 and mount it as /data +(again, this is the data from `Tulin et al., 2013 +`__). + Check:: ls /data @@ -210,8 +216,8 @@ This step can be automated with a 'for' loop at the shell prompt. Try:: for i in *.pe.fq.gz *.se.fq.gz do echo working with $i - newfile=`basename $i .fq.gz` - gunzip -c $i | fastq_quality_filter -Q33 -q 30 -p 50 | gzip -9c > ${newfile}.qc.fq.gz + newfile="$(basename $i .fq.gz)" + gunzip -c $i | fastq_quality_filter -Q33 -q 30 -p 50 | gzip -9c > "${newfile}.qc.fq.gz" done What this loop does is: @@ -302,7 +308,7 @@ compressed. Let's do some mass renaming:: for i in *.pe.qc.fq.gz.pe do - newfile=`basename $i .pe.qc.fq.gz.pe`.pe.qc.fq + newfile="$(basename $i .pe.qc.fq.gz.pe).pe.qc.fq" mv $i $newfile gzip $newfile done @@ -311,7 +317,7 @@ and also some mass combining:: for i in *.pe.qc.fq.gz.se do - otherfile=`basename $i .pe.qc.fq.gz.se`.se.qc.fq.gz + otherfile="$(basename $i .pe.qc.fq.gz.se).se.qc.fq.gz" gunzip -c $otherfile > combine cat $i >> combine gzip -c combine > $otherfile @@ -350,5 +356,5 @@ instead of /data!), then do :: which will copy all of the files over from the ./save directory onto the '/save' disk. Then 'umount /save' and voila, you've got a copy of the files! -Next stop: :doc:`2-digital-normalization`. +Next stop: :doc:`2-diginorm`. diff --git a/doc/mrnaseq/2-diginorm.txt b/doc/mrnaseq/2-diginorm.txt index af330ed..ab4154c 100644 --- a/doc/mrnaseq/2-diginorm.txt +++ b/doc/mrnaseq/2-diginorm.txt @@ -74,8 +74,8 @@ We can combine the orphaned reads into a single file:: for i in *.se.qc.fq.gz.keep.abundfilt do - pe_orphans=`basename $i .se.qc.fq.gz.keep.abundfilt`.pe.qc.fq.gz.keep.abundfilt.se - newfile=`basename $i .se.qc.fq.gz.keep.abundfilt`.se.qc.keep.abundfilt.fq.gz + pe_orphans=$(basename $i .se.qc.fq.gz.keep.abundfilt).pe.qc.fq.gz.keep.abundfilt.se + newfile=$(basename $i .se.qc.fq.gz.keep.abundfilt).se.qc.keep.abundfilt.fq.gz cat $i $pe_orphans | gzip -c > $newfile done @@ -83,12 +83,12 @@ We can also rename the remaining PE reads & compress those files:: for i in *.abundfilt.pe do - newfile=`basename $i .fq.gz.keep.abundfilt.pe`.keep.abundfilt.fq + newfile=$(basename $i .fq.gz.keep.abundfilt.pe).keep.abundfilt.fq mv $i $newfile gzip $newfile done -This leaves you with a whole passle o' files, most of which you want to go +This leaves you with a whole passel o' files, most of which you want to go away! 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz @@ -99,7 +99,7 @@ away! 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz.keep 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz.keep.abundfilt -And, finally, let's get rid of a lot of the old files :: +So, finally, let's get rid of a lot of the old files :: rm *.se.qc.fq.gz.keep.abundfilt rm *.pe.qc.fq.gz.keep.abundfilt.se @@ -115,4 +115,6 @@ You should now have:: 6Hour_CGATGT_L002_R1_005.pe.qc.keep.abundfilt.fq.gz 6Hour_CGATGT_L002_R1_005.se.qc.keep.abundfilt.fq.gz +@CTB what are these? + Save all these files to a new volume, and get ready to assemble! diff --git a/doc/mrnaseq/3-big-assembly.txt b/doc/mrnaseq/3-big-assembly.txt new file mode 100644 index 0000000..e535ff5 --- /dev/null +++ b/doc/mrnaseq/3-big-assembly.txt @@ -0,0 +1,75 @@ +============================== +3. Running the Actual Assembly +============================== + +All of the below should be run in screen, probably... You will want +at least 15 GB of RAM, maybe more. + +(If you start up a new machine, you'll need to go to +:doc:`1-quality` and install khmer and screed.) + +.. note:: + + You can start this tutorial with the contents of EC2/EBS snapshot + snap-7b0b872e. + +Installing Trinity +------------------ + +To install Trinity:: + + cd /root + + curl -L http://sourceforge.net/projects/trinityrnaseq/files/latest/download?source=files > trinity.tar.gz + + tar xzf trinity.tar.gz + cd trinityrnaseq_r2013-02-25/ + export FORCE_UNSAFE_CONFIGURE=1 + make + +Install bowtie +-------------- + +Download and install bowtie:: + + cd /root + curl -O -L http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip + unzip bowtie-0.12.7-linux-x86_64.zip + cd bowtie-0.12.7 + cp bowtie bowtie-build bowtie-inspect /usr/local/bin + +Build the files to assemble +--------------------------- + +For paired-end data, Trinity expects two files, 'left' and 'right'; +there can be orphan sequences present, however. So, below, we split +all of our interleaved pair files in two, and then add the single-ended +seqs to one of 'em. :: + + cd /mnt/work + for i in *.pe.qc.keep.abundfilt.fq.gz + do + python /usr/local/share/khmer/sandbox/split-pe.py $i + done + + cat *.1 > left.fq + cat *.2 > right.fq + + gunzip -c *.se.qc.keep.abundfilt.fq.gz >> left.fq + +Assembling with Trinity +----------------------- + +Run the assembler! :: + + /root/trinityrnaseq_r2013-02-25/Trinity.pl --left left.fq --right right.fq --seqType fq -JM 15G + +Note that this last bit (15G) is the maximum amount of memory to use. You +can increase (or decrease) it based on what machine you rented. This size +works for the m1.xlarge machines. + +Once this completes (on the Nematostella data it might take about 12 hours), +you'll have an assembled transcriptome in trinity_out_dir/Trinity.fasta. + +You can now copy it over via Dropbox, or set it up for BLAST (see +:doc:`installing-blastkit`). diff --git a/doc/mrnaseq/5-building-transcript-families.txt b/doc/mrnaseq/5-building-transcript-families.txt new file mode 100644 index 0000000..af9dde5 --- /dev/null +++ b/doc/mrnaseq/5-building-transcript-families.txt @@ -0,0 +1,97 @@ +=============================== +5. Building transcript families +=============================== + +Install khmer, screed, and BLAST. (See :doc:`1-quality` and +:doc:`installing-blastkit`). I would suggest using an m1.large or +m1.xlarge machine. + +You'll also need to install some eel-pond scripts:: + + git clone https://github.com/ctb/eel-pond.git /usr/local/share/eel-pond + +Copy in your data +================= + +You need to get ahold of your assembled transcriptome (from +e.g. :doc:`3-big-assembly`). Put it in /mnt. + +For the purposes of your first run through, I suggest just grabbing my copy +of the Nematostella assembly:: + + cd /mnt + curl -O https://s3.amazonaws.com/public.ged.msu.edu/trinity-nematostella-raw.fa.gz + +Run khmer partitioning +====================== + +Partitioning runs a de Bruijn graph-based clustering algorithm that will +cluster your transcripts by transitive sequence overlap. That is, it will +build transcript families :). :: + + /usr/local/share/khmer/scripts/do-partition.py -x 1e9 -N 4 --threads 4 nema trinity-nematostella-raw.fa.gz + +This should take about 15 minutes, and outputs a file ending in '.part' +that contains the partition assignments. Now, group and rename the +sequences:: + + python /usr/local/share/eel-pond/rename-with-partitions.py nema trinity-nematostella-raw.fa.gz.part + mv trinity-nematostella-raw.fa.gz.part.renamed.fasta.gz trinity-nematostella.renamed.fa.gz + +.. tail n.dist +.. (warning) + +.. + +.. (explain sequence names) + +Looking at the renamed sequences +================================ + +Let's look at the renamed sequences:: + + gunzip -c trinity-nematostella.renamed.fa.gz | head + +You'll see that each sequence name looks like this:: + + >nema.id1.tr16001 1_of_1_in_tr16001 len=261 id=1 tr=16001 + +Some explanation: + + - 'nema' is the prefix that you gave the rename script, above; + + - 'idN' is the unique ID for this sequence; it will never be repeated in this + file. + + - 'trN' is the transcript family, which may contain one or more transcripts. + + - '1_of_1_in_tr16001' tells you that this transcript family has only + one transcript in it (this one!) Other transcript families may + (will) have more. + + - 'len' is the sequence length. + +Doing a preliminary annotation against mouse +============================================ + +Now let's assign putative homology & orthology to these transcripts, by +doing BLASTs & reciprocal best hit analysis. First, uncompress your +transcripts file:: + + gunzip trinity-nematostella.renamed.fa.gz + +Now, grab the latest mouse RefSeq:: + + curl -O ftp://ftp.ncbi.nih.gov/refseq/M_musculus/mRNA_Prot/mouse.protein.faa.gz + gunzip mouse.protein.faa.gz + +Format both as BLAST databases:: + + formatdb -i mouse.protein.faa -o T -p T + formatdb -i trinity-nematostella.renamed.fa -o T -p F + +And, now, run BLAST in both directions. Note, this may take ~24 hours or +longer; you probably want to run it in screen:: + + blastall -i trinity-nematostella.renamed.fa -d mouse.protein.faa -e 1e-3 -p blastx -o nema.x.mouse -a 8 + blastall -i mouse.protein.faa -d trinity-nematostella.renamed.fa -e 1e-3 -p tblastn -o mouse.x.nema -a 8 diff --git a/doc/mrnaseq/index.txt b/doc/mrnaseq/index.txt index 02a6ab2..d09b6f2 100644 --- a/doc/mrnaseq/index.txt +++ b/doc/mrnaseq/index.txt @@ -11,6 +11,10 @@ Intro material: 2-diginorm 3-big-assembly + installing-blastkit + + 5-building-transcript-families + .. de-novo-assembly .. rsem From 6e07ee9d12f41a4bc0287a3cfff486bc9ed7d668 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Wed, 7 Aug 2013 23:09:41 -0400 Subject: [PATCH 05/14] foo --- .../5-building-transcript-families.txt | 35 +++++++ doc/mrnaseq/installing-blastkit.txt | 93 +++++++++++++++++++ doc/tutorials-2013/installing-dropbox.txt | 5 + 3 files changed, 133 insertions(+) create mode 100644 doc/mrnaseq/installing-blastkit.txt diff --git a/doc/mrnaseq/5-building-transcript-families.txt b/doc/mrnaseq/5-building-transcript-families.txt index af9dde5..bce3cb5 100644 --- a/doc/mrnaseq/5-building-transcript-families.txt +++ b/doc/mrnaseq/5-building-transcript-families.txt @@ -95,3 +95,38 @@ longer; you probably want to run it in screen:: blastall -i trinity-nematostella.renamed.fa -d mouse.protein.faa -e 1e-3 -p blastx -o nema.x.mouse -a 8 blastall -i mouse.protein.faa -d trinity-nematostella.renamed.fa -e 1e-3 -p tblastn -o mouse.x.nema -a 8 + +.. note:: + + You may need to do:: + + cd /usr/local/share/eel-pond + git pull origin master + + to update the source code. + +Assigning names to sequences +============================ + +Now, calculate putative homology (best BLAST hit) and orthology +(reciprocal best hits):: + + python /usr/local/share/eel-pond/make-uni-best-hits.py nema.x.mouse nema.x.mouse.homol + python /usr/local/share/eel-pond/make-reciprocal-best-hits.py nema.x.mouse mouse.x.nema nema.x.mouse.ortho + +Prepare some of the mouse info:: + + python /usr/local/share/eel-pond/make-namedb.py mouse.protein.faa mouse.namedb + python -m screed.fadbm mouse.protein.faa + +And, finally, annotate the sequences:: + + python /usr/local/share/eel-pond/annotate-seqs.py trinity-nematostella.renamed.fa nema.x.mouse.ortho nema.x.mouse.homol + +This will produce a file 'trinity-nematostella.renamed.fa.annot', which +will have sequences that look like this:: + + >nematostella.id1.tr115222 h=43% => suppressor of tumorigenicity 7 protein isoform 2 [Mus musculus] 1_of_7_in_tr115222 len=1635 id=1 tr=115222 1_of_7_in_tr115222 len=1635 id=1 tr=115222 + +I suggest renaming this file to 'nematostella.fa' and using it for +BLASTs (see :doc:`installing-blastkit`). diff --git a/doc/mrnaseq/installing-blastkit.txt b/doc/mrnaseq/installing-blastkit.txt new file mode 100644 index 0000000..75d9bb2 --- /dev/null +++ b/doc/mrnaseq/installing-blastkit.txt @@ -0,0 +1,93 @@ +============================ +BLASTing your assembled data +============================ + +One thing everyone wants to do is BLAST sequence data, right? Here's a +simple way to set up a stylish little BLAST server that lets you search +your newly assembled sequences. + +Installing blastkit +------------------- + +Installing some prerequisites:: + + pip install pygr + pip install whoosh + pip install git+https://github.com/ctb/pygr-draw.git + pip install git+https://github.com/ged-lab/screed.git + apt-get -y install lighttpd + +and configure them:: + + cd /etc/lighttpd/conf-enabled + ln -fs ../conf-available/10-cgi.conf ./ + echo 'cgi.assign = ( ".cgi" => "" )' >> 10-cgi.conf + echo 'index-file.names += ( "index.cgi" ) ' >> 10-cgi.conf + + /etc/init.d/lighttpd restart + +Next, install BLAST:: + + cd /root + + curl -O ftp://ftp.ncbi.nih.gov/blast/executables/release/2.2.24/blast-2.2.24-x64-linux.tar.gz + tar xzf blast-2.2.24-x64-linux.tar.gz + cp blast-2.2.24/bin/* /usr/local/bin + cp -r blast-2.2.24/data /usr/local/blast-data + +And put in blastkit:: + + cd /root + git clone https://github.com/ctb/blastkit.git -b ec2 + cd blastkit/www + ln -fs $PWD /var/www/blastkit + + mkdir files + chmod a+rxwt files + chmod +x /root + +and run check.py; it should say everything is OK. + +Adding the data +--------------- + +If you've just finished a transcriptome assembly (:doc:`3-big-assembly`) then +you can do this to copy your newly generated assembly into the right place:: + + cp trinity_out_dir/Trinity.fasta /root/blastkit/db/db.fa + +Alternatively, you can grab my version of the assembly (from running this +tutorial):: + + cd /root/blastkit + curl -O https://s3.amazonaws.com/public.ged.msu.edu/trinity-nematostella-raw.fa.gz + gunzip trinity-nematostella-raw.fa.gz + mv trinity-nematostella-raw.fa db/db.fa + +Formatting the database +~~~~~~~~~~~~~~~~~~~~~~~ + +After you've done either of the above, format and install the database +for blastkit:: + + cd /root/blastkit + formatdb -i db/db.fa -o T -p F + python index-db.py db/db.fa + +Done! + +.. note:: + + You can install any file of DNA sequences you want this way; just copy + it into /root/blastkit/db/db.fa and run the indexing commands, above. + +Running blastkit +---------------- + +Figure out what your machine name is +(ec2-???-???-???-???.compute-1.amazonaws.com) and go to:: + + http://machine-name/blastkit/ + +Make sure you have enabled port 80 in your security settings on Amazon. + diff --git a/doc/tutorials-2013/installing-dropbox.txt b/doc/tutorials-2013/installing-dropbox.txt index 794cceb..efa846a 100644 --- a/doc/tutorials-2013/installing-dropbox.txt +++ b/doc/tutorials-2013/installing-dropbox.txt @@ -17,6 +17,11 @@ Unpack it:: tar -xvzf dropbox.tar.gz +Make the Dropbox directory on /mnt and link it in:: + + mkdir /mnt/Dropbox + ln -fs /mnt/Dropbox /root + and then run it:: ~/.dropbox-dist/dropboxd & From 96ed29c4b88d46447a35c5b924d5214067384f67 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Fri, 9 Aug 2013 15:20:32 -0400 Subject: [PATCH 06/14] added mapping/quantitation --- doc/2013-stamps/1-quality.txt | 11 ++ doc/2013-stamps/1-setup.txt | 27 +++++ doc/2013-stamps/2-quality.txt | 80 +++++++++++++ doc/2013-stamps/3-diginorm.txt | 106 +++++++++++++++++ doc/2013-stamps/4-partition.txt | 82 +++++++++++++ doc/2013-stamps/5-assemble.txt | 110 ++++++++++++++++++ .../6-mapping-and-quantitation.txt | 87 ++++++++++++++ doc/2013-stamps/index.txt | 12 ++ .../5-building-transcript-families.txt | 4 +- doc/mrnaseq/index.txt | 2 +- 10 files changed, 518 insertions(+), 3 deletions(-) create mode 100644 doc/2013-stamps/1-quality.txt create mode 100644 doc/2013-stamps/1-setup.txt create mode 100644 doc/2013-stamps/2-quality.txt create mode 100644 doc/2013-stamps/3-diginorm.txt create mode 100644 doc/2013-stamps/4-partition.txt create mode 100644 doc/2013-stamps/5-assemble.txt create mode 100644 doc/2013-stamps/6-mapping-and-quantitation.txt create mode 100644 doc/2013-stamps/index.txt diff --git a/doc/2013-stamps/1-quality.txt b/doc/2013-stamps/1-quality.txt new file mode 100644 index 0000000..91f1d84 --- /dev/null +++ b/doc/2013-stamps/1-quality.txt @@ -0,0 +1,11 @@ +========================================= +1. Adapter trimming and quality filtering +========================================= + +Configure your environment:: + + echo 'export PYTHONPATH=$PYTHONPATH:/class/stamps-software/share/khmer/python' >> ~/.bash_login + echo 'export PYTHONPATH=$PYTHONPATH:/class/stamps-software/share/khmer/python' >> ~/.bashrc + echo 'export PATH=/class/stamps-software/bin:$PATH' >> ~/.bashrc + echo 'export PATH=/class/stamps-software/bin:$PATH' >> ~/.bash_login + diff --git a/doc/2013-stamps/1-setup.txt b/doc/2013-stamps/1-setup.txt new file mode 100644 index 0000000..9fd5434 --- /dev/null +++ b/doc/2013-stamps/1-setup.txt @@ -0,0 +1,27 @@ +======== +1. Setup +======== + +Configure your environment:: + + echo 'export PYTHONPATH=$PYTHONPATH:/class/stamps-software/share/khmer/python' >> ~/.bash_login + echo 'export PYTHONPATH=$PYTHONPATH:/class/stamps-software/share/khmer/python' >> ~/.bashrc + echo 'export PATH=/class/stamps-software/bin:$PATH' >> ~/.bashrc + echo 'export PATH=/class/stamps-software/bin:$PATH' >> ~/.bash_login + + source ~/.bashrc + +Create a virtual environment and activate it:: + + python -m virtualenv ~/env + . ~/env/bin/activate + +Install screed in your virtual environment:: + + pip install git+https://github.com/ged-lab/screed.git + +Also see: :doc:`../mrnaseq/using-screen`. + +---- + +Next: :doc:`2-quality`. diff --git a/doc/2013-stamps/2-quality.txt b/doc/2013-stamps/2-quality.txt new file mode 100644 index 0000000..b258e97 --- /dev/null +++ b/doc/2013-stamps/2-quality.txt @@ -0,0 +1,80 @@ +========================================= +2. Adapter trimming and quality filtering +========================================= + +.. note:: + + Activate your virtual environment and make sure you're running in screen! (see :doc:`../mrnaseq/using-screen`) + +.. note:: + + The finished (QC'ed) files for this are in /class/cbrown/data/SRR49206*.qc.fq.gz + +Create a working directory +========================== + +Let's create a place to work:: + + cd + mkdir assembly + cd assembly + +Trim and quality filter +======================= + +Grab some Illumina adapters:: + + curl -O https://s3.amazonaws.com/public.ged.msu.edu/illuminaClipping.fa + +Trim the first data set (~20 minutes):: + + mkdir trim + cd trim + + java -jar /class/stamps-software/share/Trimmomatic-0.27/trimmomatic-0.27.jar PE /class/cbrown/data/SRR492065_?.fastq.gz s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 + + python /class/stamps-software/share/khmer/sandbox/interleave.py s?_pe > combined.fq + + fastq_quality_filter -Q33 -q 30 -p 50 -i combined.fq > combined-trim.fq + fastq_quality_filter -Q33 -q 30 -p 50 -i s1_se > s1_se.trim + python /class/stamps-software/share/khmer/sandbox/strip-and-split-for-assembly.py combined-trim.fq + + gzip -9c combined-trim.fq.pe > ../SRR492065.pe.qc.fq.gz + gzip -9c combined-trim.fq.se s1_se > ../SRR492065.se.qc.fq.gz + + cd ../ + rm -fr trim + +Trim the second data set (~20 minutes):: + + mkdir trim + cd trim + + java -jar /class/stamps-software/share/Trimmomatic-0.27/trimmomatic-0.27.jar PE /class/cbrown/data/SRR492066_?.fastq.gz s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 + + python /class/stamps-software/share/khmer/sandbox/interleave.py s?_pe > combined.fq + + fastq_quality_filter -Q33 -q 30 -p 50 -i combined.fq > combined-trim.fq + fastq_quality_filter -Q33 -q 30 -p 50 -i s1_se > s1_se.trim + python /class/stamps-software/share/khmer/sandbox/strip-and-split-for-assembly.py combined-trim.fq + + gzip -9c combined-trim.fq.pe > ../SRR492066.pe.qc.fq.gz + gzip -9c combined-trim.fq.se s1_se > ../SRR492066.se.qc.fq.gz + + cd ../ + rm -fr trim + +Done! Now you have four files: SRR492065.pe.qc.fq.gz, SRR492065.se.qc.fq.gz, SRR492066.pe.qc.fq.gz, and SRR492066.se.qc.fq.gz. + +The '.pe' files are interleaved paired-end; you can take a look at them like so:: + + gunzip -c SRR492065.pe.qc.fq.gz | head + +The other two are single-ended files, where the reads have been +orphaned because we discarded stuff. + +All four files are in FASTQ format. + +---- + +Next: :doc:`3-diginorm` diff --git a/doc/2013-stamps/3-diginorm.txt b/doc/2013-stamps/3-diginorm.txt new file mode 100644 index 0000000..407d8e6 --- /dev/null +++ b/doc/2013-stamps/3-diginorm.txt @@ -0,0 +1,106 @@ +================================ +3. Running digital normalization +================================ + +.. note:: + + Activate your virtual environment and make sure you're running in screen! + +Start with the QC'ed files from :doc:`2-quality` or copy them into a +working directory:: + + cd + mkdir assembly + cp /class/cbrown/data/SRR49206*.qc.fq.gz . + +Run a first round of digital normalization +========================================== + +Normalize everything to a coverage of 20, starting with the (more valuable) +PE reads; keep pairs using '-p':: + + python /class/stamps-software/share/khmer/scripts/normalize-by-median.py -k 20 -C 20 -N 4 -x 5e8 -p --savehash normC20k20.kh *.pe.qc.fq.gz + +...and continuing into the (less valuable but maybe still useful) SE reads:: + + python /class/stamps-software/share/khmer/scripts/normalize-by-median.py -C 20 --savehash normC20k20.kh --loadhash normC20k20.kh *.se.qc.fq.gz + +This produces a set of '.keep' files, as well as a normC20k20.kh +database file. + +Error-trim your data +==================== + +Use 'filter-abund' to trim off any k-mers that are abundance-1 in +high-coverage reads (-V option, for variable coverage):: + + /class/stamps-software/share/khmer/scripts/filter-abund.py -V normC20k20.kh *.keep + +This produces .abundfilt files. + +The process of error trimming could have orphaned reads, so split the +PE file into still-interleaved and non-interleaved reads:: + + for i in *.pe.qc.fq.gz.keep.abundfilt + do + python /class/stamps-software/share/khmer/sandbox/strip-and-split-for-assembly.py $i + done + +This leaves you with PE files (.pe.qc.fq.gz.keep.abundfilt.pe :) and +two sets of SE files (.se.qc.fq.gz.keep.abundfilt and +.pe.qc.fq.gz.keep.abundfilt.se). Yes, I did indeed devise this naming +scheme. + +Normalize down to C=5 +===================== + +Now that we've eliminated many more erroneous k-mers, let's ditch some more +high-coverage data. First, normalize the paired-end reads:: + + /class/stamps-software/share/khmer/scripts/normalize-by-median.py -C 5 -k 20 -N 4 -x 5e8 --savehash normC5k20.kh -p *.pe.qc.fq.gz.keep.abundfilt.pe + +and then do the remaining single-ended reads:: + + /class/stamps-software/share/khmer/scripts/normalize-by-median.py -C 5 --savehash normC5k20.kh --loadhash normC5k20.kh *.pe.qc.fq.gz.keep.abundfilt.se *.se.qc.fq.gz.keep.abundfilt + +Compress and combine the files +============================== + +Now let's tidy things up. Here are the paired files (kak = +keep/abundfilt/keep):: + + gzip -9c SRR492065.pe.qc.fq.gz.keep.abundfilt.pe.keep > SRR492065.pe.kak.qc.fq.gz + gzip -9c SRR492066.pe.qc.fq.gz.keep.abundfilt.pe.keep > SRR492066.pe.kak.qc.fq.gz + +and the single-ended files:: + + gzip -9c SRR492066.pe.qc.fq.gz.keep.abundfilt.se.keep SRR492066.se.qc.fq.gz.keep.abundfilt.keep > SRR492066.se.kak.qc.fq.gz + gzip -9c SRR492065.pe.qc.fq.gz.keep.abundfilt.se.keep SRR492065.se.qc.fq.gz.keep.abundfilt.keep > SRR492065.se.kak.qc.fq.gz + +Read stats +========== + +Try running:: + + python /class/stamps-software/share/khmer/sandbox/readstats.py *.kak.qc.fq.gz *.?e.qc.fq.gz + +after a long wait, you'll see :: + + --------------- + 861769600 bp / 8617696 seqs; 100.0 average length -- SRR492065.pe.qc.fq.gz + 79586148 bp / 802158 seqs; 99.2 average length -- SRR492065.se.qc.fq.gz + 531691400 bp / 5316914 seqs; 100.0 average length -- SRR492066.pe.qc.fq.gz + 89903689 bp / 904157 seqs; 99.4 average length -- SRR492066.se.qc.fq.gz + + 173748898 bp / 1830478 seqs; 94.9 average length -- SRR492065.pe.kak.qc.fq.gz + 8825611 bp / 92997 seqs; 94.9 average length -- SRR492065.se.kak.qc.fq.gz + 52345833 bp / 550900 seqs; 95.0 average length -- SRR492066.pe.kak.qc.fq.gz + 10280721 bp / 105478 seqs; 97.5 average length -- SRR492066.se.kak.qc.fq.gz + + --------------- + +Not bad -- considerably more than 80% of the reads were eliminated in the kak! + +---- + +Next: :doc:`4-partition` diff --git a/doc/2013-stamps/4-partition.txt b/doc/2013-stamps/4-partition.txt new file mode 100644 index 0000000..f56a16f --- /dev/null +++ b/doc/2013-stamps/4-partition.txt @@ -0,0 +1,82 @@ +=============== +4. Partitioning +=============== + +.. note:: + + Activate your virtual environment and make sure you're running in screen! + +Start with the QC'ed files from :doc:`2-quality` or copy them into a +working directory:: + + cd + mkdir assembly + cp /class/cbrown/data/SRR49206*.kak.qc.fq.gz . + +Simple partitioning +=================== + +Partitioning is a rather complex process -- nowhere near as nice and +simple as digital normalization. However, we do have a simple script +to run the basic stuff; if this script is too slow, or doesn't work +well for big chunks of data, we might have remedies. But for the +meantime, try the simple script:: + + python /class/stamps-software/share/khmer/scripts/do-partition.py -k 32 -x 1e9 --threads 4 kak *.kak.qc.fq.gz + +This should take about 15 minutes, and will produce '.part' files. These +are now FASTA files that contain partition annotations. For example, check +out:: + + head SRR492065.pe.kak.qc.fq.gz + +Extracting the partitions into groups +===================================== + +Generally there are *lots* of partitions, and for convenience sake we +group them into group files that can be assembled in small chunks. +To do this, :: + + python /class/stamps-software/share/khmer/scripts/extract-partitions.py -X 100000 kak *.part + +This will leave you with a bunch of 'kak.group*.fa', as well as a '.dist' +file containing the distribution of partition sizes (how many sequences are +in a given partition). + +Here, the '-X' sets the number of sequences stuck into a group file. +By default the -X parameter is 1 million, which would put all of the +sequences into a single file for this data set. + +Occasionally (OK, rather frequently) you'll find that almost all of +your sequences coalesce into one partition, which we unaffectionately +call the 'lump'. There are many possible reasons for this, and we +have a series of increasingly large hammers that can be used on the +lump. + +For now, simply observe that:: + + tail kak.dist + +reports that about 2/3 of the sequences are in a single partition:: + + 1674746 1 112164 2539252 + +Reinflating partitions (optional) +================================= + +At this point it's worth noting that the partitions are *normalized*, +that is, diginormed. That makes it hard to use them for abundance +calculations, and some assemblers prefer to have the original +abundances in there. + +So, ran you recover the abundances? Of course you can! However, you +do have to combine all of the raw (unpartitioned) reads into a single +file, because the script to reinflate the partitions takes only single +file. Sorry :(. :: + + gunzip -c /class/cbrown/data/SRR49206?.?e.qc.fq.gz > all.fq + python /class/stamps-software/share/khmer/sandbox/sweep-reads3.py -x 1e8 kak.group*.fa all.fq + +---- + +Next: :doc:`5-assemble` diff --git a/doc/2013-stamps/5-assemble.txt b/doc/2013-stamps/5-assemble.txt new file mode 100644 index 0000000..1b07dfd --- /dev/null +++ b/doc/2013-stamps/5-assemble.txt @@ -0,0 +1,110 @@ +============= +5. Assembling +============= + +At last! All that filtering and diginorming is done, and we can get +down to the serious business of assembling. Huzzah! + +.. note:: + + You can copy the finished products from :doc:`4-partition` into your + directory from /class/cbrown/data if you like:: + + cd + mkdir assembly + cp /class/cbrown/data/group*.fa*gz + +Splitting out PE and SE reads +============================= + +Let's assemble just one group, for now -- group 5 should be nice and +small. Generally assemblers will want interleaved reads to be +distinguished from orphaned reads, so let's split 'em out:: + + mkdir group5 + cd group5 + python /class/stamps-software/share/khmer/sandbox/strip-and-split-for-assembly.py ../kak.group0005.fa.gz + python ~/khmer/sandbox/strip-and-split-for-assembly.py ../kak.group0005.fa.gz.sweep3 + mv kak.group0005.fa.gz.sweep3.pe kak.group5.nodn.pe + mv kak.group0005.fa.gz.sweep3.se kak.group5.nodn.se + +Using Velvet +============ + +I personally really like the Velvet assembler, since it yields pretty +good results in a wide variety of situations. It's also rather fast. +The downside is that you have to specify a 'k' parameter, which gets +annoying because it gives you different results (see presentation). + +So, let's just assemble across a lot of k's. :: + + for k in {21..43..2} + do + /class/stamps-software/bin/velveth dn.$k $k -fasta -shortPaired kak.group0005.fa.gz.pe -short kak.group0005.fa.gz.se + /class/stamps-software/bin/velvetg dn.$k -exp_cov auto + done + + for k in {21..43..2} + do + /class/stamps-software/bin/velveth nodn.$k $k -fasta -shortPaired kak.group5.nodn.pe -short kak.group5.nodn.se + /class/stamps-software/bin/velvetg nodn.$k -exp_cov auto + done + + +Using IDBA +========== + +I've heard good things about IDBA. Let's give it a try:: + + /class/stamps-software/bin/idba_ud --pre_correction -r kak.group0005.fa.gz.pe -o idba.dn.d + + /class/stamps-software/bin/idba_ud --pre_correction -r kak.group5.nodn.pe -o idba.nodn.d/ + +Getting stats for the assemblies +================================ + +To get some basic stats for the assemblies, run:: + + python /class/stamps-software/share/khmer/sandbox/assemstats3.py 500 *.??/contigs.fa idba.*.d/scaffold.fa + +This will yield something like:: + + N sum max filename + 38 671957 83467 dn.21/contigs.fa + 32 668918 83568 dn.23/contigs.fa + 35 668509 83401 dn.25/contigs.fa + 31 671843 83817 dn.27/contigs.fa + 32 669104 83721 dn.29/contigs.fa + 32 672735 84066 dn.31/contigs.fa + 32 673102 83774 dn.33/contigs.fa + 31 674629 83912 dn.35/contigs.fa + 31 677446 84200 dn.37/contigs.fa + 33 681099 84554 dn.39/contigs.fa + 35 685245 84852 dn.41/contigs.fa + 40 686733 85276 dn.43/contigs.fa + 41 649574 62719 nodn.21/contigs.fa + 39 639388 62155 nodn.23/contigs.fa + 49 646132 62145 nodn.25/contigs.fa + 39 647100 83798 nodn.27/contigs.fa + 38 650487 83750 nodn.29/contigs.fa + 33 649863 83770 nodn.31/contigs.fa + 31 636979 83822 nodn.33/contigs.fa + 35 645536 83856 nodn.35/contigs.fa + 36 647848 83800 nodn.37/contigs.fa + 33 654660 83934 nodn.39/contigs.fa + 36 645126 83897 nodn.41/contigs.fa + 34 660289 83231 idba.dn.d/scaffold.fa + 45 666147 41120 idba.nodn.d/scaffold.fa + +Extracting sequences over a certain length +========================================== + +Let's say that we want to work with the sequences in dn.43/contigs.fa, but we want to get rid of all the small sequences. How? + +Easy:: + + /class/stamps-software/share/khmer/sandbox/extract-long-sequences.py 500 dn.43/contigs.fa > group5-assembly.fa + +---- + +Next: :doc:`6-mapping-and-quantitation`. diff --git a/doc/2013-stamps/6-mapping-and-quantitation.txt b/doc/2013-stamps/6-mapping-and-quantitation.txt new file mode 100644 index 0000000..d786889 --- /dev/null +++ b/doc/2013-stamps/6-mapping-and-quantitation.txt @@ -0,0 +1,87 @@ +===================================== +6. Mapping and abundance quantitation +===================================== + +Let's do some simple mapping to do abundance estimation in the group 5 +assembly. + +Setup +===== + +First, move to a new directory:: + + cd + mkdir mapping + cd mapping + + cp /class/cbrown/data/group5-assembly.fa.gz . + + gunzip group5-assembly.fa + +Bowtie mapping +============== + +`bowtie `__ is a commonly +used mapping tool. + +We need to build a bowtie reference:: + + bowtie-build group5-assembly.fa group5 + +and then let's map a subset of the reads (make it the single-ended ones, +just to keep things fast):: + + gunzip -c ~/data/SRR492065.se.qc.fq.gz | \ + bowtie -q group5 - > SRR492065.x.group5.map + gunzip -c ~/data/SRR492066.se.qc.fq.gz | \ + bowtie -q group5 - > SRR492066.x.group5.map + +Here you should see the following output for the first:: + + # reads processed: 802158 + # reads with at least one reported alignment: 63226 (7.88%) + # reads that failed to align: 738932 (92.12%) + Reported 63226 alignments to 1 output stream(s) + +and the second:: + + # reads processed: 904157 + # reads with at least one reported alignment: 39572 (4.38%) + # reads that failed to align: 864585 (95.62%) + Reported 39572 alignments to 1 output stream(s) + +At the moment, there seems to be no good way to do automated differential +analysis of two samples, so I'll content myself with showing you how to +annotate the assembled sequences with the mapping abundances. + +To do this, we will need to make two copies of the annotated assembly +-- one annotated with the first (SRR492065) and the other with the +second (SRR492066) abundances. :: + + python /class/stamps-software/share/khmer/sandbox/make-coverage.py group5-assembly.fa SRR492065.x.group5.map + mv group5-assembly.fa.cov group5.SRR492065.fa + + python /class/stamps-software/share/khmer/sandbox/make-coverage.py group5-assembly.fa SRR492066.x.group5.map + mv group5-assembly.fa.cov group5.SRR492066.fa + +What you should see now is that there's a [cov] annotation for each +sequence in every file -- :: + + head -1 group5.SRR49206?.fa + +and you should see something like this -- + + ==> group5.SRR492065.fa <== + >NODE_1_length_3642_cov_7.200439[cov=1] + + ==> group5.SRR492066.fa <== + >NODE_1_length_3642_cov_7.200439[cov=2] + +This format can be uploaded directly to MG-RAST as an +abundance-annotated assembly, although there's no good way to do +comparative analysis yet, it seems. + +More generally, I would suggest looking into things like `RSEM +`__, `DEseq +`__, or other +differential expression packages for mRNAseq. diff --git a/doc/2013-stamps/index.txt b/doc/2013-stamps/index.txt new file mode 100644 index 0000000..85c6118 --- /dev/null +++ b/doc/2013-stamps/index.txt @@ -0,0 +1,12 @@ +The STAMPS Metagenome Assembly Tutorial +======================================= + +.. toctree:: + :maxdepth: 1 + + 1-setup + 2-quality + 3-diginorm + 4-partition + 5-assemble + 6-mapping-and-quantitation diff --git a/doc/mrnaseq/5-building-transcript-families.txt b/doc/mrnaseq/5-building-transcript-families.txt index bce3cb5..b562113 100644 --- a/doc/mrnaseq/5-building-transcript-families.txt +++ b/doc/mrnaseq/5-building-transcript-families.txt @@ -93,8 +93,8 @@ Format both as BLAST databases:: And, now, run BLAST in both directions. Note, this may take ~24 hours or longer; you probably want to run it in screen:: - blastall -i trinity-nematostella.renamed.fa -d mouse.protein.faa -e 1e-3 -p blastx -o nema.x.mouse -a 8 - blastall -i mouse.protein.faa -d trinity-nematostella.renamed.fa -e 1e-3 -p tblastn -o mouse.x.nema -a 8 + blastall -i trinity-nematostella.renamed.fa -d mouse.protein.faa -e 1e-3 -p blastx -o nema.x.mouse -a 8 -v 4 -b 4 + blastall -i mouse.protein.faa -d trinity-nematostella.renamed.fa -e 1e-3 -p tblastn -o mouse.x.nema -a 8 -v 4 -b 4 .. note:: diff --git a/doc/mrnaseq/index.txt b/doc/mrnaseq/index.txt index d09b6f2..a2bae09 100644 --- a/doc/mrnaseq/index.txt +++ b/doc/mrnaseq/index.txt @@ -1,7 +1,7 @@ The Eel Pond mRNAseq Tutorial ============================= -Intro material: +The tutorial: .. toctree:: :maxdepth: 1 From b285215a905e888c6438779480edf9c1c1452f3b Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Mon, 26 Aug 2013 21:55:07 -0400 Subject: [PATCH 07/14] last commit before rm --- doc/2013-stamps/4-partition.txt | 4 ++-- doc/2013-stamps/5-assemble.txt | 2 +- doc/mrnaseq/4-diff-expr.txt | 4 ++++ doc/mrnaseq/5-building-transcript-families.txt | 15 +++------------ doc/mrnaseq/installing-blastkit.txt | 7 ++++++- doc/mrnaseq/notes | 14 ++++++++++++++ 6 files changed, 30 insertions(+), 16 deletions(-) create mode 100644 doc/mrnaseq/4-diff-expr.txt create mode 100644 doc/mrnaseq/notes diff --git a/doc/2013-stamps/4-partition.txt b/doc/2013-stamps/4-partition.txt index f56a16f..adbf5d7 100644 --- a/doc/2013-stamps/4-partition.txt +++ b/doc/2013-stamps/4-partition.txt @@ -6,7 +6,7 @@ Activate your virtual environment and make sure you're running in screen! -Start with the QC'ed files from :doc:`2-quality` or copy them into a +Start with the QC'ed files from :doc:`3-diginorm` or copy them into a working directory:: cd @@ -28,7 +28,7 @@ This should take about 15 minutes, and will produce '.part' files. These are now FASTA files that contain partition annotations. For example, check out:: - head SRR492065.pe.kak.qc.fq.gz + head SRR492065.pe.kak.qc.fq.gz.part Extracting the partitions into groups ===================================== diff --git a/doc/2013-stamps/5-assemble.txt b/doc/2013-stamps/5-assemble.txt index 1b07dfd..aec7b7a 100644 --- a/doc/2013-stamps/5-assemble.txt +++ b/doc/2013-stamps/5-assemble.txt @@ -24,7 +24,7 @@ distinguished from orphaned reads, so let's split 'em out:: mkdir group5 cd group5 python /class/stamps-software/share/khmer/sandbox/strip-and-split-for-assembly.py ../kak.group0005.fa.gz - python ~/khmer/sandbox/strip-and-split-for-assembly.py ../kak.group0005.fa.gz.sweep3 + python /class/stamps-software/share/khmer/sandbox/strip-and-split-for-assembly.py ../kak.group0005.fa.gz.sweep3 mv kak.group0005.fa.gz.sweep3.pe kak.group5.nodn.pe mv kak.group0005.fa.gz.sweep3.se kak.group5.nodn.se diff --git a/doc/mrnaseq/4-diff-expr.txt b/doc/mrnaseq/4-diff-expr.txt new file mode 100644 index 0000000..f173b6a --- /dev/null +++ b/doc/mrnaseq/4-diff-expr.txt @@ -0,0 +1,4 @@ +curl -O http://athyra.idyll.org/~t/petMar_lamp3.longest.fasta.gz +gunzip petMar_lamp3.longest.fasta.gz + +rsem-prepare-reference petMar_lamp3.longest.fasta lamp3 diff --git a/doc/mrnaseq/5-building-transcript-families.txt b/doc/mrnaseq/5-building-transcript-families.txt index b562113..793b3e8 100644 --- a/doc/mrnaseq/5-building-transcript-families.txt +++ b/doc/mrnaseq/5-building-transcript-families.txt @@ -1,6 +1,6 @@ -=============================== -5. Building transcript families -=============================== +============================================================ +5. Building transcript families and annotating the sequences +============================================================ Install khmer, screed, and BLAST. (See :doc:`1-quality` and :doc:`installing-blastkit`). I would suggest using an m1.large or @@ -96,15 +96,6 @@ longer; you probably want to run it in screen:: blastall -i trinity-nematostella.renamed.fa -d mouse.protein.faa -e 1e-3 -p blastx -o nema.x.mouse -a 8 -v 4 -b 4 blastall -i mouse.protein.faa -d trinity-nematostella.renamed.fa -e 1e-3 -p tblastn -o mouse.x.nema -a 8 -v 4 -b 4 -.. note:: - - You may need to do:: - - cd /usr/local/share/eel-pond - git pull origin master - - to update the source code. - Assigning names to sequences ============================ diff --git a/doc/mrnaseq/installing-blastkit.txt b/doc/mrnaseq/installing-blastkit.txt index 75d9bb2..162be62 100644 --- a/doc/mrnaseq/installing-blastkit.txt +++ b/doc/mrnaseq/installing-blastkit.txt @@ -46,7 +46,12 @@ And put in blastkit:: chmod a+rxwt files chmod +x /root -and run check.py; it should say everything is OK. +and run check.py:: + + cd /root/blastkit + python ./check.py + +It should say everything is OK. Adding the data --------------- diff --git a/doc/mrnaseq/notes b/doc/mrnaseq/notes new file mode 100644 index 0000000..7e0dc7a --- /dev/null +++ b/doc/mrnaseq/notes @@ -0,0 +1,14 @@ +apt-get -y install r-cran-gplots +cd /root/rsem-1.2.5 + +make ebseq + +cd /mnt + + +time rsem-calculate-expression -p 4 --paired-end 1_SC_1_week_ATCACG_L002_R1_001.pe.qc.fq.gz.1 1_SC_1_week_ATCACG_L002_R1_001.pe.qc.fq.gz.2 lamp3 2_BR_1_week_pe + +rsem-generate-data-matrix 2_BR_1_week_se.genes.results 1_SC_1_week_se.genes.results > 1_week.matrix + 116 more 1_week.matrix + 117 rsem-find-DE 1_week.matrix 1 0.5 xxx + From a6667dfeb9d02306707761a379b0b69357042f01 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Mon, 26 Aug 2013 21:58:54 -0400 Subject: [PATCH 08/14] updated stamps to point at protocols --- doc/2013-stamps/index.txt | 3 + doc/mrnaseq/0-download-and-save.txt | 90 ----- doc/mrnaseq/1-quality.txt | 360 ------------------ doc/mrnaseq/2-diginorm.txt | 120 ------ doc/mrnaseq/3-big-assembly.txt | 75 ---- doc/mrnaseq/4-diff-expr.txt | 4 - .../5-building-transcript-families.txt | 123 ------ doc/mrnaseq/coral.txt | 7 - doc/mrnaseq/de-novo-assembly.txt | 144 ------- doc/mrnaseq/images/AttachVolume.png | Bin 51196 -> 0 bytes doc/mrnaseq/images/AttachVolumeWindow.png | Bin 45750 -> 0 bytes doc/mrnaseq/images/ClickVolumes.png | Bin 83673 -> 0 bytes doc/mrnaseq/images/CreateVolume.png | Bin 84340 -> 0 bytes doc/mrnaseq/images/CreateVolumeWindow.png | Bin 32117 -> 0 bytes doc/mrnaseq/index.txt | 18 +- doc/mrnaseq/installing-blastkit.txt | 98 ----- doc/mrnaseq/notes | 14 - doc/mrnaseq/rsem.txt | 96 ----- doc/mrnaseq/saving-data-persistently.txt | 114 ------ doc/mrnaseq/using-screen.txt | 58 --- 20 files changed, 4 insertions(+), 1320 deletions(-) delete mode 100644 doc/mrnaseq/0-download-and-save.txt delete mode 100644 doc/mrnaseq/1-quality.txt delete mode 100644 doc/mrnaseq/2-diginorm.txt delete mode 100644 doc/mrnaseq/3-big-assembly.txt delete mode 100644 doc/mrnaseq/4-diff-expr.txt delete mode 100644 doc/mrnaseq/5-building-transcript-families.txt delete mode 100644 doc/mrnaseq/coral.txt delete mode 100644 doc/mrnaseq/de-novo-assembly.txt delete mode 100644 doc/mrnaseq/images/AttachVolume.png delete mode 100644 doc/mrnaseq/images/AttachVolumeWindow.png delete mode 100644 doc/mrnaseq/images/ClickVolumes.png delete mode 100644 doc/mrnaseq/images/CreateVolume.png delete mode 100644 doc/mrnaseq/images/CreateVolumeWindow.png delete mode 100644 doc/mrnaseq/installing-blastkit.txt delete mode 100644 doc/mrnaseq/notes delete mode 100644 doc/mrnaseq/rsem.txt delete mode 100644 doc/mrnaseq/saving-data-persistently.txt delete mode 100644 doc/mrnaseq/using-screen.txt diff --git a/doc/2013-stamps/index.txt b/doc/2013-stamps/index.txt index 85c6118..f47aceb 100644 --- a/doc/2013-stamps/index.txt +++ b/doc/2013-stamps/index.txt @@ -1,6 +1,9 @@ The STAMPS Metagenome Assembly Tutorial ======================================= +**Note:** This tutorial is being updated and maintained over at +`the khmer protocols `__. + .. toctree:: :maxdepth: 1 diff --git a/doc/mrnaseq/0-download-and-save.txt b/doc/mrnaseq/0-download-and-save.txt deleted file mode 100644 index 1a407bf..0000000 --- a/doc/mrnaseq/0-download-and-save.txt +++ /dev/null @@ -1,90 +0,0 @@ -=========================================== -0. Downloading and Saving Your Initial Data -=========================================== - -We're going to run transcriptome assembly completely in the cloud, -because that way (a) you don't need to buy a big computer, and (b) -I don't have to figure out all the special details of your own -computer system. - -This does mean that the first thing you need to do is get your data -over to the cloud. I tend to just store it there in the first place, -because... - -The basics ----------- - -...Amazon is happy to rent disk space to you, in addition to compute time. -They'll rent you disk space in a few different ways, but the way that's -most useful for us is through what's called Elastic Block Store. This -is essentially a hard-disk rental service. - -There are two basic concepts -- "volume" and "snapshot". A "volume" can -be thought of as a pluggable-in hard drive: you create an empty volume of -a given size, attach it to a running instance, and voila! You have extra -hard disk space. Volume-based hard disks have two problems, however: -first, they cannot be used outside of the "availability zone" they've -been created in, which means that you need to be careful to put them -in the same zone that your instance is running in; and they can't be shared -amongst people. - -Snapshots, the second concept, are the solution to transporting and -sharing the data on volumes. A "snapshot" is essentially a frozen -copy of your volume; you can copy a volume into a snapshot, and a -snapshot into a volume. - -Getting started ---------------- - -Run through :doc:`saving-data-persistently` once, to get the hang of -the mechanics. Essentially you create a disk; attach it; format it; -and then copy things to and from it. - -Downloading and saving your data to a volume --------------------------------------------- - -There are *many* different ways of getting big sequence files to and -from Amazon. The two that I mostly use are 'curl', which downloads -files from a Web site URL; and 'ncftp', which is a robust FTP client -that let's you get files from an FTP site. Sequencing centers almost -always make their data available in one of these two ways. - -.. note:: - - To use ncftp on your Amazon instance, you may need to install it:: - - apt-get -y install ncftp - -For example, to retrieve a file from an FTP site, you would do something -like:: - - cd /mnt - ncftp -u ftp://path/to/FTP/site - -use 'cd' to find the right directory, and then:: - - >> mget * - -to download the files. Then type 'quit'. You can also use 'curl' to -download files one at a time from Web or FTP sites. - -Once you have the files, figure out their size using 'du -sk' (e.g. after the -above, 'du -sk /mnt' will tell you how much data you have saved under /mnt), -and go create and attach a volume (see :doc:`saving-data-persistently`). - -This data is now something that will *stick around* when you shut down -your instance. It's a good rule of thumb to do "savepoints" -- whenever -you complete a big chunk of work, think about saving the data at that -point. I've broken the mRNAseq tutorial down into chunks of work where -you can do this -- after each Web page, basically. - -Some test data --------------- - -To get started with multfile analysis and assembly, I've provided some -test mRNAseq data from embryonic stages of *Nematostella vectensis*; -the source is `this excellent paper -`__ by Tulin et al., "A -quantitative reference transcriptome for Nematostella vectensis". The -data is on snapshot 'snap-f5a9dea7', so go create a volume from that -and mount it as '/data' to get started. diff --git a/doc/mrnaseq/1-quality.txt b/doc/mrnaseq/1-quality.txt deleted file mode 100644 index 9b52b96..0000000 --- a/doc/mrnaseq/1-quality.txt +++ /dev/null @@ -1,360 +0,0 @@ -================================================ -1. Quality Trimming and Filtering Your Sequences -================================================ - -Boot up an m1.xlarge machine from Amazon Web Services; this has about -15 GB of RAM, and 2 CPUs, and will be enough to complete the assembly -of the Nematostella data set. - -.. note:: - - This follows the NGS 2013 tutorial, - :doc:`../tutorials-2013/short-read-quality-evaluation`, but for multiple - files. - -.. note:: - - The end results of this tutorial are available as public snapshot - snap-8b155fd9 on EC2/EBS. - -Install software ----------------- - -Install screed:: - - cd /usr/local/share - git clone https://github.com/ged-lab/screed.git - cd screed - python setup.py install - -Install the bleeding-edge version of khmer:: - - cd /usr/local/share - git clone https://github.com/ged-lab/khmer.git -b bleeding-edge - cd khmer - make - - echo 'export PYTHONPATH=/usr/local/share/khmer/python' >> ~/.bashrc - source ~/.bashrc - -Install Trimmomatic:: - - cd /root - curl -O http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/Trimmomatic-0.27.zip - unzip Trimmomatic-0.27.zip - cp Trimmomatic-0.27/trimmomatic-0.27.jar /usr/local/bin - -Install libgtextutils and fastx:: - - cd /root - curl -O http://hannonlab.cshl.edu/fastx_toolkit/libgtextutils-0.6.1.tar.bz2 - tar xjf libgtextutils-0.6.1.tar.bz2 - cd libgtextutils-0.6.1/ - ./configure && make && make install - - cd /root - curl -O http://hannonlab.cshl.edu/fastx_toolkit/fastx_toolkit-0.0.13.2.tar.bz2 - tar xjf fastx_toolkit-0.0.13.2.tar.bz2 - cd fastx_toolkit-0.0.13.2/ - ./configure && make && make install - -In each of these cases, we're downloading the software -- you can use -google to figure out what each package is and does if we don't discuss -it below. We're then unpacking it, sometimes compiling it (which we -can discuss later), and then installing it for general use. - -Find your data --------------- - -Either load in your own data (as in :doc:`0-download-and-save`) or -create a volume from snapshot snap-f5a9dea7 and mount it as /data -(again, this is the data from `Tulin et al., 2013 -`__). - -Check:: - - ls /data - -If you see all the files you think you should, good! Otherwise, debug. - -If you're using the Tulin et al. data provided in the snapshot above, -you should see:: - - /data/0Hour_ATCACG_L002_R1_001.fastq.gz - -Link your data into a working directory ---------------------------------------- - -Rather than *copying* the files into the working directory, let's just -*link* them in -- this creates a reference so that UNIX knows where to -find them but doesn't need to actually move them around. :: - - cd /mnt - mkdir work - cd work - - ln -fs /data/*.fastq.gz . - -(The 'ln' command is what does the linking.) - -Download the Illumina adapters ------------------------------- - -In the working directory, :: - - curl -O https://s3.amazonaws.com/public.ged.msu.edu/illuminaClipping.fa - -.. note:: - - You'll need to make sure these are the right adapters for your data. - If they are, you should see that some of them are trimmed off, below; - if they're not, you shouldn't see anything get trimmed. - -Adapter trim each pair of files -------------------------------- - -(From this point on, you may want to be running things inside of -screen, so that you detach and log out while it's running; see -:doc:`using-screen` for more information.) - -If you're following along using the Nematostella data, you should have a -bunch of files that look like this (use 'ls' to show them):: - - 24HourB_GCCAAT_L002_R1_001.fastq.gz - ^^ - -Each file with an R1 in its name should have a matching file with an R2 -- -these are the paired ends. - -.. note:: - - You'll need to replace and , below, with the - names of your actual R1 and R2 files. You'll also need to replace - with something that's unique to each pair of files. - It doesn't really matter what, but you need to make sure it's different - for each pair of files. - -For *each* of these pairs, run the following:: - - # make a temp directory - mkdir trim - cd trim - - # run trimmomatic - java -jar /usr/local/bin/trimmomatic-0.27.jar PE s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 - - # interleave the remaining paired-end files - python /usr/local/share/khmer/sandbox/interleave.py s1_pe s2_pe | gzip -9c > ../.pe.fq.gz - - # combine the single-ended files - cat s1_se s2_se | gzip -9c > ../.se.fq.gz - - # go back up to the working directory and remove the temp directory - cd .. - rm -r trim - - # make it hard to delete the files you just created - chmod u-w *.pe.fq.gz *.se.fq.gz - -You can read the '#' comments above, but, briefly, this set of commands: - - * creates a temporary directory, 'trim/' - - * runs 'Trimmomatic' in that directory to trim off the adapters, and then - puts remaining pairs (most of them!) in s1_pe and s2_pe, and any orphaned - singletons in s1_se and s2_se. - - * interleaves the paired ends and puts them back in the working directory - - * combines the orphaned reads and puts them back in the working directory - -Automating things a bit -~~~~~~~~~~~~~~~~~~~~~~~ - -OK, once you've done this once or twice, it gets kind of tedious, doesn't it? -I've written a script to write these commands out automatically. Run it -like so:: - - cd /mnt/work - python /usr/local/share/khmer/sandbox/write-trimmomatic.py > trim.sh - -Run this, and then look at 'trim.sh' using the 'more' command -- :: - - more trim.sh - -If it looks like it contains the right commands, you can run it by doing:: - - bash trim.sh - -.. note:: - - This is a prime example of scripting to make your life much easier - and less error prone. Take a look at this file sometime -- - 'more /usr/local/share/khmer/sandbox/write-trimmomatic.py' to get - some idea of how this works. - -Quality trim each pair of files -------------------------------- - -After you run this, you should have a bunch of '*.pe.fq.gz' files and -a bunch of '*.se.fq.gz' files. The former are files that contain paired, -interleaved sequences; the latter contain single-ended, non-interleaved -sequences. - -Next, for each of these files, run:: - - gunzip -c | fastq_quality_filter -Q33 -q 30 -p 50 | gzip -9c > .qc.fq.gz - -This uncompresses each file, removes poor-quality sequences, and then -recompresses it. Note that (following :doc:`../tutorials-2013/short-read-quality-evaluation`) you can also trim things by putting in a 'fastx_trimmer -Q33 -l 70 |' into the mix. - -Automating this step -~~~~~~~~~~~~~~~~~~~~ - -This step can be automated with a 'for' loop at the shell prompt. Try:: - - for i in *.pe.fq.gz *.se.fq.gz - do - echo working with $i - newfile="$(basename $i .fq.gz)" - gunzip -c $i | fastq_quality_filter -Q33 -q 30 -p 50 | gzip -9c > "${newfile}.qc.fq.gz" - done - -What this loop does is: - - - for every file ending in pe.fq.gz and se.fq.gz, - - - print out a message with the filename, - - - construct a name 'newfile' that omits the trailing .fq.gz - - - uncompresses the original file, passes it through fastq, recompresses it, - and saves it as "newfile".qc.fq.gz - -Extracting paired ends from the interleaved files -------------------------------------------------- - -The fastx utilities that we're using to do quality trimming aren't -paired-end aware; they're removing individual sequences. Because the -pe files are interleaved, this means that there may now be some orphaned -sequences in there. Downstream, we will want to pay special attention -to the remaining paired sequences, so we want to separate out the pe -and se files. How do we go about that? Another script, of course! - -The khmer script 'strip-and-split-for-assembly.py' does exactly that. -You run it on an interleaved file that may have some orphans, and it -produces .pe and .se files afterwards, containing pairs and orphans -respectively. - -To run it on all of the pe qc files, do:: - - for i in *.pe*.qc.fq.gz - do - python /usr/local/share/khmer/sandbox/strip-and-split-for-assembly.py $i - done - -Finishing up ------------- - -You should now have a whole mess of files. For example, in the Nematostella -data, for *each* of the original input files, you'll have:: - - 24HourB_GCCAAT_L002_R1_001.fastq.gz - the original data - 24HourB_GCCAAT_L002_R2_001.fastq.gz - 24HourB_GCCAAT_L002_R1_001.pe.fq.gz - adapter trimmed pe - 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz - FASTX filtered - 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.pe - FASTX filtered PE - 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.se - FASTX filtered SE - 24HourB_GCCAAT_L002_R1_001.se.fq.gz - adapter trimmed orphans - 24HourB_GCCAAT_L002_R1_001.se.qc.fq.gz - FASTX filtered orphans - -Yikes! What to do? - -Well, first, you can get rid of the original data. You already have it on a -disk somewhere, right? :: - - rm *.fastq.gz - -Next, you can get rid of the 'pe.fq.gz' and 'se.fq.gz' files, since you -only want the QC files. So:: - - rm *.pe.fq.gz *.se.fq.gz - -And, finally, you can toss the pe.fq.gz files, because you've turned *those* -into .pe and .se files. - - rm *.pe.qc.fq.gz - -So now you should be left with only three files for each sample:: - - 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.pe - FASTX filtered PE - 24HourB_GCCAAT_L002_R1_001.pe.qc.fq.gz.se - FASTX filtered SE - 24HourB_GCCAAT_L002_R1_001.se.qc.fq.gz - FASTX filtered orphans - -Things to think about -~~~~~~~~~~~~~~~~~~~~~ - -Note that the filenames, while ugly, are conveniently structured with the -history of what you've done. This is a good idea. - -Also note that we've conveniently named the files so that we can remove -the unwanted ones en masse. This is a good idea, too. - -Renaming files --------------- - -I'm a fan of keeping the files named somewhat sensibly, and keeping them -compressed. Let's do some mass renaming:: - - for i in *.pe.qc.fq.gz.pe - do - newfile="$(basename $i .pe.qc.fq.gz.pe).pe.qc.fq" - mv $i $newfile - gzip $newfile - done - -and also some mass combining:: - - for i in *.pe.qc.fq.gz.se - do - otherfile="$(basename $i .pe.qc.fq.gz.se).se.qc.fq.gz" - gunzip -c $otherfile > combine - cat $i >> combine - gzip -c combine > $otherfile - rm $i - done - -and finally:: - - chmod u-w *.qc.fq.gz - -to make sure you don't accidentally delete something. - -Saving the files ----------------- - -At this point, you should save these files, which will be used in two -ways: first, for assembly; and second, for mapping, to do quantitation -and ultimately comparative expression analysis. You can save them by -doing this:: - - mkdir save - mv *.qc.fq.gz save - du -sk save - -This puts the data you want to save into a subdirectory named 'save', and -calculates the size. - -Now, create a volume of the given size -- divide by a thousand to get -gigabytes, multiply by 1.1 to make sure you have enough room, and then -follow the instructions in :doc:`saving-data-persistently`. Once -you've mounted it properly (I would suggest mounting it on /save -instead of /data!), then do :: - - rsync -av save /save - -which will copy all of the files over from the ./save directory onto the -'/save' disk. Then 'umount /save' and voila, you've got a copy of the files! - -Next stop: :doc:`2-diginorm`. - diff --git a/doc/mrnaseq/2-diginorm.txt b/doc/mrnaseq/2-diginorm.txt deleted file mode 100644 index ab4154c..0000000 --- a/doc/mrnaseq/2-diginorm.txt +++ /dev/null @@ -1,120 +0,0 @@ -================================= -2. Applying Digital Normalization -================================= - -.. note:: - - You can start this tutorial with the contents of EC2/EBS snapshot - snap-126cc847. - -.. note:: - - You'll need ~15 GB of RAM for this, or more if you have a LOT of data. - -Link in your data ------------------ - -Make sure your data is in /mnt/work/. If you've loaded it onto /data, -you can do:: - - cd /mnt - mkdir work - cd /mnt/work - ln -fs /data/*.qc.fq.gz . - -Run digital normalization -------------------------- - -Apply digital normalization to the paired-end reads:: - - python /usr/local/share/khmer/scripts/normalize-by-median.py -p -k 20 -C 20 -N 4 -x 3e9 --savehash normC20k20.kh *.pe.qc.fq.gz - -and then to the single-end reads:: - - python /usr/local/share/khmer/scripts/normalize-by-median.py -k 20 -C 20 -N 4 -x 3e9 --loadhash normC20k20.kh --savehash normC20k20.kh *.se.qc.fq.gz - -Note the '-p' in the first normalize-by-median command -- when run on -PE data, that ensures that no paired ends are orphaned. However, it -will complain on single-ended data, so you have to give the data to it -separately. - -Also note the '-N' and '-x' parameters. These specify how much memory -diginorm should use. The product of these should be less than the -memory size of the machine you selected. The maximum needed for *any* -transcriptome should be in the ~60 GB range, e.g. -N 4 -x 15e9; for -only a few hundred million reads, 16 GB should be plenty. (See -`choosing hash sizes for khmer -`__ -for more information.) - -Trim off likely erroneous k-mers -------------------------------- - -Now, run through all the reads and trim off low-abundance parts of -high-coverage reads:: - - /usr/local/share/khmer/scripts/filter-abund.py -V normC20k20.kh *.keep - -This will turn some reads into orphans, but that's ok -- their partner -read was bad. - -Rename files -~~~~~~~~~~~~ - -You'll have a bunch of 'keep.abundfilt' files -- let's make things prettier. - -First, let's break out the orphaned and still-paired reads:: - - for i in *.pe.*.abundfilt; - do - python /usr/local/share/khmer/sandbox/strip-and-split-for-assembly.py $i - done - -We can combine the orphaned reads into a single file:: - - for i in *.se.qc.fq.gz.keep.abundfilt - do - pe_orphans=$(basename $i .se.qc.fq.gz.keep.abundfilt).pe.qc.fq.gz.keep.abundfilt.se - newfile=$(basename $i .se.qc.fq.gz.keep.abundfilt).se.qc.keep.abundfilt.fq.gz - cat $i $pe_orphans | gzip -c > $newfile - done - -We can also rename the remaining PE reads & compress those files:: - - for i in *.abundfilt.pe - do - newfile=$(basename $i .fq.gz.keep.abundfilt.pe).keep.abundfilt.fq - mv $i $newfile - gzip $newfile - done - -This leaves you with a whole passel o' files, most of which you want to go -away! - - 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz - 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz.keep - 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz.keep.abundfilt - 6Hour_CGATGT_L002_R1_005.pe.qc.fq.gz.keep.abundfilt.se - 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz - 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz.keep - 6Hour_CGATGT_L002_R1_005.se.qc.fq.gz.keep.abundfilt - -So, finally, let's get rid of a lot of the old files :: - - rm *.se.qc.fq.gz.keep.abundfilt - rm *.pe.qc.fq.gz.keep.abundfilt.se - rm *.keep - rm *.abundfilt - rm *.qc.fq.gz - -Gut check -~~~~~~~~~ - -You should now have:: - - 6Hour_CGATGT_L002_R1_005.pe.qc.keep.abundfilt.fq.gz - 6Hour_CGATGT_L002_R1_005.se.qc.keep.abundfilt.fq.gz - -@CTB what are these? - -Save all these files to a new volume, and get ready to assemble! diff --git a/doc/mrnaseq/3-big-assembly.txt b/doc/mrnaseq/3-big-assembly.txt deleted file mode 100644 index e535ff5..0000000 --- a/doc/mrnaseq/3-big-assembly.txt +++ /dev/null @@ -1,75 +0,0 @@ -============================== -3. Running the Actual Assembly -============================== - -All of the below should be run in screen, probably... You will want -at least 15 GB of RAM, maybe more. - -(If you start up a new machine, you'll need to go to -:doc:`1-quality` and install khmer and screed.) - -.. note:: - - You can start this tutorial with the contents of EC2/EBS snapshot - snap-7b0b872e. - -Installing Trinity ------------------- - -To install Trinity:: - - cd /root - - curl -L http://sourceforge.net/projects/trinityrnaseq/files/latest/download?source=files > trinity.tar.gz - - tar xzf trinity.tar.gz - cd trinityrnaseq_r2013-02-25/ - export FORCE_UNSAFE_CONFIGURE=1 - make - -Install bowtie --------------- - -Download and install bowtie:: - - cd /root - curl -O -L http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip - unzip bowtie-0.12.7-linux-x86_64.zip - cd bowtie-0.12.7 - cp bowtie bowtie-build bowtie-inspect /usr/local/bin - -Build the files to assemble ---------------------------- - -For paired-end data, Trinity expects two files, 'left' and 'right'; -there can be orphan sequences present, however. So, below, we split -all of our interleaved pair files in two, and then add the single-ended -seqs to one of 'em. :: - - cd /mnt/work - for i in *.pe.qc.keep.abundfilt.fq.gz - do - python /usr/local/share/khmer/sandbox/split-pe.py $i - done - - cat *.1 > left.fq - cat *.2 > right.fq - - gunzip -c *.se.qc.keep.abundfilt.fq.gz >> left.fq - -Assembling with Trinity ------------------------ - -Run the assembler! :: - - /root/trinityrnaseq_r2013-02-25/Trinity.pl --left left.fq --right right.fq --seqType fq -JM 15G - -Note that this last bit (15G) is the maximum amount of memory to use. You -can increase (or decrease) it based on what machine you rented. This size -works for the m1.xlarge machines. - -Once this completes (on the Nematostella data it might take about 12 hours), -you'll have an assembled transcriptome in trinity_out_dir/Trinity.fasta. - -You can now copy it over via Dropbox, or set it up for BLAST (see -:doc:`installing-blastkit`). diff --git a/doc/mrnaseq/4-diff-expr.txt b/doc/mrnaseq/4-diff-expr.txt deleted file mode 100644 index f173b6a..0000000 --- a/doc/mrnaseq/4-diff-expr.txt +++ /dev/null @@ -1,4 +0,0 @@ -curl -O http://athyra.idyll.org/~t/petMar_lamp3.longest.fasta.gz -gunzip petMar_lamp3.longest.fasta.gz - -rsem-prepare-reference petMar_lamp3.longest.fasta lamp3 diff --git a/doc/mrnaseq/5-building-transcript-families.txt b/doc/mrnaseq/5-building-transcript-families.txt deleted file mode 100644 index 793b3e8..0000000 --- a/doc/mrnaseq/5-building-transcript-families.txt +++ /dev/null @@ -1,123 +0,0 @@ -============================================================ -5. Building transcript families and annotating the sequences -============================================================ - -Install khmer, screed, and BLAST. (See :doc:`1-quality` and -:doc:`installing-blastkit`). I would suggest using an m1.large or -m1.xlarge machine. - -You'll also need to install some eel-pond scripts:: - - git clone https://github.com/ctb/eel-pond.git /usr/local/share/eel-pond - -Copy in your data -================= - -You need to get ahold of your assembled transcriptome (from -e.g. :doc:`3-big-assembly`). Put it in /mnt. - -For the purposes of your first run through, I suggest just grabbing my copy -of the Nematostella assembly:: - - cd /mnt - curl -O https://s3.amazonaws.com/public.ged.msu.edu/trinity-nematostella-raw.fa.gz - -Run khmer partitioning -====================== - -Partitioning runs a de Bruijn graph-based clustering algorithm that will -cluster your transcripts by transitive sequence overlap. That is, it will -build transcript families :). :: - - /usr/local/share/khmer/scripts/do-partition.py -x 1e9 -N 4 --threads 4 nema trinity-nematostella-raw.fa.gz - -This should take about 15 minutes, and outputs a file ending in '.part' -that contains the partition assignments. Now, group and rename the -sequences:: - - python /usr/local/share/eel-pond/rename-with-partitions.py nema trinity-nematostella-raw.fa.gz.part - mv trinity-nematostella-raw.fa.gz.part.renamed.fasta.gz trinity-nematostella.renamed.fa.gz - -.. tail n.dist -.. (warning) - -.. - -.. (explain sequence names) - -Looking at the renamed sequences -================================ - -Let's look at the renamed sequences:: - - gunzip -c trinity-nematostella.renamed.fa.gz | head - -You'll see that each sequence name looks like this:: - - >nema.id1.tr16001 1_of_1_in_tr16001 len=261 id=1 tr=16001 - -Some explanation: - - - 'nema' is the prefix that you gave the rename script, above; - - - 'idN' is the unique ID for this sequence; it will never be repeated in this - file. - - - 'trN' is the transcript family, which may contain one or more transcripts. - - - '1_of_1_in_tr16001' tells you that this transcript family has only - one transcript in it (this one!) Other transcript families may - (will) have more. - - - 'len' is the sequence length. - -Doing a preliminary annotation against mouse -============================================ - -Now let's assign putative homology & orthology to these transcripts, by -doing BLASTs & reciprocal best hit analysis. First, uncompress your -transcripts file:: - - gunzip trinity-nematostella.renamed.fa.gz - -Now, grab the latest mouse RefSeq:: - - curl -O ftp://ftp.ncbi.nih.gov/refseq/M_musculus/mRNA_Prot/mouse.protein.faa.gz - gunzip mouse.protein.faa.gz - -Format both as BLAST databases:: - - formatdb -i mouse.protein.faa -o T -p T - formatdb -i trinity-nematostella.renamed.fa -o T -p F - -And, now, run BLAST in both directions. Note, this may take ~24 hours or -longer; you probably want to run it in screen:: - - blastall -i trinity-nematostella.renamed.fa -d mouse.protein.faa -e 1e-3 -p blastx -o nema.x.mouse -a 8 -v 4 -b 4 - blastall -i mouse.protein.faa -d trinity-nematostella.renamed.fa -e 1e-3 -p tblastn -o mouse.x.nema -a 8 -v 4 -b 4 - -Assigning names to sequences -============================ - -Now, calculate putative homology (best BLAST hit) and orthology -(reciprocal best hits):: - - python /usr/local/share/eel-pond/make-uni-best-hits.py nema.x.mouse nema.x.mouse.homol - python /usr/local/share/eel-pond/make-reciprocal-best-hits.py nema.x.mouse mouse.x.nema nema.x.mouse.ortho - -Prepare some of the mouse info:: - - python /usr/local/share/eel-pond/make-namedb.py mouse.protein.faa mouse.namedb - python -m screed.fadbm mouse.protein.faa - -And, finally, annotate the sequences:: - - python /usr/local/share/eel-pond/annotate-seqs.py trinity-nematostella.renamed.fa nema.x.mouse.ortho nema.x.mouse.homol - -This will produce a file 'trinity-nematostella.renamed.fa.annot', which -will have sequences that look like this:: - - >nematostella.id1.tr115222 h=43% => suppressor of tumorigenicity 7 protein isoform 2 [Mus musculus] 1_of_7_in_tr115222 len=1635 id=1 tr=115222 1_of_7_in_tr115222 len=1635 id=1 tr=115222 - -I suggest renaming this file to 'nematostella.fa' and using it for -BLASTs (see :doc:`installing-blastkit`). diff --git a/doc/mrnaseq/coral.txt b/doc/mrnaseq/coral.txt deleted file mode 100644 index 673378e..0000000 --- a/doc/mrnaseq/coral.txt +++ /dev/null @@ -1,7 +0,0 @@ - -java -jar /usr/local/bin/trimmomatic-0.27.jar PE P_astreoides_larva_GCCAAT_L005_R?_001.fastq.gz s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 - - -mkdir larva -root@ip-10-40-10-213:/mnt/two# mv s?_?e larva/ -root@ip-10-40-10-213:/mnt/two# java -jar /usr/local/bin/trimmomatic-0.27.jar PE P_astreoides_settled*_L005_R?_001.fastq.gz s1_pe s1_se s2_pe s2_se ILLUMINACLIP:../illuminaClipping.fa:2:30:10 diff --git a/doc/mrnaseq/de-novo-assembly.txt b/doc/mrnaseq/de-novo-assembly.txt deleted file mode 100644 index 31e3382..0000000 --- a/doc/mrnaseq/de-novo-assembly.txt +++ /dev/null @@ -1,144 +0,0 @@ -=================================================== -Doing a small de novo mRNAseq assembly with Trinity -=================================================== - -Installing Trinity ------------------- - -To install Trinity:: - - cd /root - - curl -L http://sourceforge.net/projects/trinityrnaseq/files/latest/download?source=files > trinity.tar.gz - - tar xzf trinity.tar.gz - cd trinityrnaseq_r2013-02-25/ - export FORCE_UNSAFE_CONFIGURE=1 - make - -Install ctb Python packages ---------------------------- - -Install screed and khmer:: - - cd /usr/local/share - git clone https://github.com/ged-lab/screed.git - cd screed - python setup.py install - - cd /usr/local/share - git clone https://github.com/ged-lab/khmer.git - cd khmer - make - - echo export PYTHONPATH=/usr/local/share/khmer/python >> ~/.bashrc - source ~/.bashrc - -Installing blastkit -------------------- - -Installing some prerequisites:: - - pip install pygr - apt-get -y install lighttpd - -and configure them:: - - cd /etc/lighttpd/conf-enabled - ln -fs ../conf-available/10-cgi.conf ./ - echo 'cgi.assign = ( ".cgi" => "" )' >> 10-cgi.conf - echo 'index-file.names += ( "index.cgi" ) ' >> 10-cgi.conf - - /etc/init.d/lighttpd restart - -Next, install BLAST:: - - cd /root - - curl -O ftp://ftp.ncbi.nih.gov/blast/executables/release/2.2.24/blast-2.2.24-x64-linux.tar.gz - tar xzf blast-2.2.24-x64-linux.tar.gz - cp blast-2.2.24/bin/* /usr/local/bin - cp -r blast-2.2.24/data /usr/local/blast-data - -And put in blastkit:: - - cd /root - git clone https://github.com/ctb/blastkit.git -b ec2 - cd blastkit/www - ln -fs $PWD /var/www/blastkit - - mkdir files - chmod a+rxwt files - chmod +x /root - -Install bowtie --------------- - -Download and install bowtie:: - - cd /root - curl -O -L http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip - unzip bowtie-0.12.7-linux-x86_64.zip - cd bowtie-0.12.7 - cp bowtie bowtie-build bowtie-inspect /usr/local/bin - -Download and preparing the test data ------------------------------------- - -Grab the coral data:: - - cd /mnt - curl -O https://s3.amazonaws.com/public.ged.msu.edu/coral-settled-400k-pe.fq.gz - -Break the interleaved FASTQ data into left and right files for Trinity:: - - python /usr/local/share/khmer/sandbox/split-pe.py coral-settled-400k-pe.fq.gz - -Assemble! :: - - /root/trinityrnaseq_r2013-02-25/Trinity.pl --left coral-settled-400k-pe.fq.gz.1 --right coral-settled-400k-pe.fq.gz.2 --seqType fq -JM 5G - -Note that this last bit (5G) is the maximum amount of memory to use. You -can increase (or decrease) it based on what machine you rented. - -Are you impatient?? -~~~~~~~~~~~~~~~~~~~ - -Note: I have a little test database that you can install below INSTEAD of -doing the assembly:: - - mkdir /root/blastkit/db - curl https://s3.amazonaws.com/public.ged.msu.edu/coral-mini-assembly.fa.gz | gunzip > /root/blastkit/db/db.fa - -Set up a BLAST database ------------------------ - -Assuming everything is successful, let's make this BLASTable. - -Copy the assembly:: - - mkdir /root/blastkit/db - cp trinity_out_dir/Trinity.fasta /root/blastkit/db/db.fa - -Format the database for BLASTing:: - - cd /root/blastkit/db - formatdb -i db.fa -o T -p F - -Format the database for sequence retrieval:: - - python ../index-db.py db.fa - -Now, go to 'http:///blastkit/' and you should have a simple -BLAST interface. If you're using the coral dataset, above, try the following -query:: - - MSRADPGKNSEPSESKMSLELRPTAPSDLGRSNEAFQDEDLERQNTPGNSTVRNRVVQSGEQGHAKQDDRQITIEQEPLG - NKEDPEDDSEDEHQKGFLERKYDTICEFCRKHRVVLRSTIWAVLLTGFLALVIAACAINFHRALPLFVITLVTIFFVIWD - HLMAKYEQRIDDFLSPGRRLLDRHWFWLKWVVWSSLILAIILWLSLDTAKLGQQNLVSFGGLIMYLILLFLFSKHPTRVY - WRPVFWGIGLQFLLGLLILRTRPGFVAFDWMGRQVQTFLGYTDTGARFVFGEKYTDHFFAFKILPIVVFFSTVMSMLYYL - GLMQWIIRKVGWLMLVTMGSSPIESVVAAGNIFIGQTESPLLVQPYLPHVTKSELHTIMTAGFATIAGSVLGAYISFGVS - STHLLTASVMSAPAALAVAKLFWPETEKPKITLKSAMKMENGDSRNLLEAASQGASSSIPLVANIAANLIAFLALLSFVN - SALSWFGSMFNYPELSFELICSYIFMPFSFMMGVDWQDSFMVAKLIGYKTFFNEFVAYDHLSKLINLRKAAGPKFVNGVQ - QYMSIRSETIATYALCGFANFGSLGIVIGGLTSIAPSRKRDIASGAMRALIAGTIACFMTACIAGILSDTPVDINCHHVL - ENGRVLSNTTEVVSCCQNLFNSTVAKGPNDVVPGGNFSLYALKSCCNLLKPPTLNCNWIPNKL diff --git a/doc/mrnaseq/images/AttachVolume.png b/doc/mrnaseq/images/AttachVolume.png deleted file mode 100644 index 922a1ea545c1c7f06470ff699cecbfff9f553d7d..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 51196 zcmY(qV_;@Wvo@S$V%xT@$;7sG$F?!CZQHhO+qP{^jBoaS&UwD~{9N6&s;hc+)hb*a zCNC=n2aOF41Ox;pAug;41O#FQ1O)s868!H-=#DxE5D+@OxsZ^&gpd%Syo0TYxs@>x zkSp+#y0N;7TuY8uJlA4gM@I)v;V+RuA(xQGe1Bg;5M*R%ahm)<-|$3`@I*++P~ZGO zKalwTt(Tse_ilpg&)+?pZrNVPEi02crZ)f<%g<#HU+)w|+{>6;P(ngR?#25=^pLIr zbNEYGeqeHbU|k2$mRda-M@Pfkp5~ndATBIV68HQIpR6Z71o<|Pz&2o@w1EMEYkeDF z#%uq&XiYmaP9R9sNU?(c0J&SxNOQqGjKI5bsHZmwf^5B&9!w}7DDIJUMI0bf1R!A} zP{C3RphD!@Op^I2m}Md8Z;W8B*)0l0AZi!PiZ(Zt-Rv)TZZT3q!c)+ge)Zlze2=c5 z5aftj-$e=hw{I9VT3}ngz8A1koJKff++fdwW6FvorpxCn1f`(&Or2O&3XR(>$3WXqwIOf6JQIEpG#3-@mqufS6>w(w&F2%Y9=m~p> zpXD)8@rV47MW_lM71${`lp(2soza|8R01qS{^V`vqATE(lCZ*0`J;={05Pk4#fBBUZTCv=l#7OR(#lpGf+7q<{N7jn;T z^lglD@^y-Hiiu2@4wcThgav>C$N_8sjJ03&GPv*~Aq%}(!$Akk_D!uio7^@jxN`KO zHvHGJ+2T6F+mgJpUs?XC{wab9`?MmkBKRUGLy$vik_M8zlGc(S#vsN>#=nkGkJ!<& zCF92Rq&Pwm!R2_(_=JJ*0bJ_oxz+L{)llS&qS>ZckD6j-|Xfbq?j|Zez1bG zcC((bKAQ!bvzwKf8=G01&&;9E@fK<43eDfmVa>iSXcu^hb4awv%}CZr+z8}I?(p-; z_6+n+=JxIO#SYu$?9mPIl0X#D7Z4mc4~s?Kq{&MQN~23FNEb=Zq;)edFw(GeFqJUz z8D;AW)I!#&*ALb!*TU9WHPqK-8^D>+8C@BFj*E^I_Z?3pPb`l$k4KxT89wXJ82b!& z4+~7>jqS}S^v;dc^^NtH&0zHbhJQ}F^$yJ#4$AUuO3{EC5+ z!`%g{{**^oc~YuY(oxt`yOVD#xK~J$O3KwJZqFUgJI?>DX)TbYl$EPf-CfvMwO6fB zbSI)Dzo7O|ee6O9RV81AUWI2(cIA3Sk5@QNmBpH+uWg}?vz5{Q;I8Ga;mUZcO{2ekIjyylx>wUkt34hl7*P1p1Iw;(TLUv*J4xOpOJ}mj46%fhz*X_ z%LsfDcG6*TJL4fEB$cnZS3Ok|72w&N*cjPx+0w81Q*#OM*}PoeZNg(oqKBftU?`>U zG2FcTdqj4Ge#53uXOnmPE9pl{;Cw)#FPlBh(eAv!e=E5$Z zxZ%q*hf9eQB{M^7mi{-rGkpjsi{MZFjiuhERp$Yj4Z0ocE$soG6|W_sC6bMUh>~*C z=9BQ0a<%A{@|Jd&>}NFs>zoW;KCeGswy)POX|F2pc<&ppI|zi(c2H&rh47CsmoNq} zTo|nAw78kLorw9U4kSh-iPjL$|oQ* za3U}wC89oIa}i~cB17#%PlNWu+M$|}E0GLRzQ|fA?#OK@3}o~a&kE!V?Ov%IN1 z>&mU#t`ecPf}^_CgL8^Zx$x|H-mYbddAG8M7r8SaO{%Hm_go<}5#XoJ}nB*S*}2vc5ZVSmIb98ct4T9nEj3c21@m zCR2P6KkR%2Z1&Fbr}t;$m*y|UT4FA8`g!r_b>AZ2GoOP#rLMvk#}ib#mgd+-nvVYL z{t2iV+bQiv^TBx8{t!ACnGOCNzMq-R8R@CEVYX4V9kcplm0>&c4gca*o6tPbs@YId zyV7IZ@_j^jj_9F&D|Rb45;r%uY2SNi&q$Pbn*u|)ryE#80n}3t-1`&AH{_LAVukPH z{suPHyFU>Y*pN9FNO3RK26{`ZmWVl)(+EDCfXx?vBvh~4up^W@v_YIcL5F-|NF_l| zfmzW_o@0)`5#9*_Qydd7)gra0;l3fQ?%hGyLDZ4Vq2&JX7X2>iK@Cb7S`~T=ogQ@z zSq4c8H6}SUF)}ecftSKjLsbi&?pdc>gh**lOhPVA3@op{aJ@h^&l@nHwxA&;qp2RE zkfQ!9e=}0X@J0Y@0#_169z`KjEK@Q`KDnbFsTr*)qb|46+45=BV$*YGcPQ=|JRWvV zTf|ZXL*}=hG#u5JH54$R)GO7mIW{uLu~{?N!=OhON~}&kNY2VkOYz}7sCTHiD8MQc zDPF0Q>FDVsD7)YFz(DUQzTPG544?$i4DCspS%_+Qz0EG^rqQlMHXv7XnEFf|_3UM) zBz$-3ofPZE3Ed^dixo|@luay7d{Q%CE35uil~MUFA6kN1y4d;Co-!}*@vU+Y6Y?l? 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Here's a -simple way to set up a stylish little BLAST server that lets you search -your newly assembled sequences. - -Installing blastkit -------------------- - -Installing some prerequisites:: - - pip install pygr - pip install whoosh - pip install git+https://github.com/ctb/pygr-draw.git - pip install git+https://github.com/ged-lab/screed.git - apt-get -y install lighttpd - -and configure them:: - - cd /etc/lighttpd/conf-enabled - ln -fs ../conf-available/10-cgi.conf ./ - echo 'cgi.assign = ( ".cgi" => "" )' >> 10-cgi.conf - echo 'index-file.names += ( "index.cgi" ) ' >> 10-cgi.conf - - /etc/init.d/lighttpd restart - -Next, install BLAST:: - - cd /root - - curl -O ftp://ftp.ncbi.nih.gov/blast/executables/release/2.2.24/blast-2.2.24-x64-linux.tar.gz - tar xzf blast-2.2.24-x64-linux.tar.gz - cp blast-2.2.24/bin/* /usr/local/bin - cp -r blast-2.2.24/data /usr/local/blast-data - -And put in blastkit:: - - cd /root - git clone https://github.com/ctb/blastkit.git -b ec2 - cd blastkit/www - ln -fs $PWD /var/www/blastkit - - mkdir files - chmod a+rxwt files - chmod +x /root - -and run check.py:: - - cd /root/blastkit - python ./check.py - -It should say everything is OK. - -Adding the data ---------------- - -If you've just finished a transcriptome assembly (:doc:`3-big-assembly`) then -you can do this to copy your newly generated assembly into the right place:: - - cp trinity_out_dir/Trinity.fasta /root/blastkit/db/db.fa - -Alternatively, you can grab my version of the assembly (from running this -tutorial):: - - cd /root/blastkit - curl -O https://s3.amazonaws.com/public.ged.msu.edu/trinity-nematostella-raw.fa.gz - gunzip trinity-nematostella-raw.fa.gz - mv trinity-nematostella-raw.fa db/db.fa - -Formatting the database -~~~~~~~~~~~~~~~~~~~~~~~ - -After you've done either of the above, format and install the database -for blastkit:: - - cd /root/blastkit - formatdb -i db/db.fa -o T -p F - python index-db.py db/db.fa - -Done! - -.. note:: - - You can install any file of DNA sequences you want this way; just copy - it into /root/blastkit/db/db.fa and run the indexing commands, above. - -Running blastkit ----------------- - -Figure out what your machine name is -(ec2-???-???-???-???.compute-1.amazonaws.com) and go to:: - - http://machine-name/blastkit/ - -Make sure you have enabled port 80 in your security settings on Amazon. - diff --git a/doc/mrnaseq/notes b/doc/mrnaseq/notes deleted file mode 100644 index 7e0dc7a..0000000 --- a/doc/mrnaseq/notes +++ /dev/null @@ -1,14 +0,0 @@ -apt-get -y install r-cran-gplots -cd /root/rsem-1.2.5 - -make ebseq - -cd /mnt - - -time rsem-calculate-expression -p 4 --paired-end 1_SC_1_week_ATCACG_L002_R1_001.pe.qc.fq.gz.1 1_SC_1_week_ATCACG_L002_R1_001.pe.qc.fq.gz.2 lamp3 2_BR_1_week_pe - -rsem-generate-data-matrix 2_BR_1_week_se.genes.results 1_SC_1_week_se.genes.results > 1_week.matrix - 116 more 1_week.matrix - 117 rsem-find-DE 1_week.matrix 1 0.5 xxx - diff --git a/doc/mrnaseq/rsem.txt b/doc/mrnaseq/rsem.txt deleted file mode 100644 index 4d4dbe1..0000000 --- a/doc/mrnaseq/rsem.txt +++ /dev/null @@ -1,96 +0,0 @@ -=================== -Expression analysis -=================== - -Installing rsem ---------------- - -We'll be using the `RSEM package `__ -to do some expression analysis. To install it:: - - cd /root - - curl -O http://deweylab.biostat.wisc.edu/rsem/src/rsem-1.2.5.tar.gz - - tar xzvf rsem-1.2.5.tar.gz - cd rsem-1.2.5 - make - -And now add this directory into your PATH, which is where Unix looks for -things to run:: - - echo 'export PATH=$PATH:/root/rsem-1.2.5' >> /root/.bashrc - source /root/.bashrc - -Installing bowtie ------------------ - -If you didn't install bowtie on this machine already (e.g. as part of -:doc:`de-novo-assembly`), RSEM needs it; do:: - - cd /root - curl -O -L http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip - unzip bowtie-0.12.7-linux-x86_64.zip - cd bowtie-0.12.7 - cp bowtie bowtie-build bowtie-inspect /usr/local/bin - -Running rsem ------------- - -Go to a working directory on /mnt:: - - cd /mnt - mkdir rsem - cd rsem - -Now, grab the reference transcriptome:: - - curl https://s3.amazonaws.com/public.ged.msu.edu/coral-mini-assembly.fa.gz | gunzip > coral.fa - - -and some reads:: - - curl -O http://athyra.idyll.org/~t/larva-ct-800k.fq.gz - curl -O http://athyra.idyll.org/~t/settled-ct-800k.fq.gz - -You'll need to uncompress the reads:: - - gunzip *.gz - -Now, prepare the reference for RSEM:: - - rsem-prepare-reference coral.fa coral - -Here, 'coral.fa' can be one of several files (comma-separated) in case -you have multiple different reference data sets. In this case it's -the transcriptome you generated, but there are lots of options (see -`the docs for rsem-prepare-reference -`__). - -And, finally, calculate the expression levels for the 'settled' and 'larva' -data sets:: - - rsem-calculate-expression settled-ct-800k.fq coral settled - rsem-calculate-expression larva-ct-800k.fq coral larva - -(See `the rsem-calculate-expression docs `__ for more info.) - -This outputs a couple files; the two most interesting, for now, are -'larva.genes.results' and 'settled.genes.results'. These contain the -following data:: - - head larva.genes.results - -should show you :: - - gene_id transcript_id(s) length effective_length expected_count TPM FPKM - comp0_c0_seq1 comp0_c0_seq1 291.00 291.00 3.00 102.12 164.47 - ... - -If you've installed Dropbox (see -:doc:`../tutorials-2013/installing-dropbox`), you can now copy the -files to Dropbox:: - - cp larva.genes.results settled.genes.results ~/Dropbox/ - -and you can open them directly in Excel as tab-delimited files. diff --git a/doc/mrnaseq/saving-data-persistently.txt b/doc/mrnaseq/saving-data-persistently.txt deleted file mode 100644 index 6fc8ab5..0000000 --- a/doc/mrnaseq/saving-data-persistently.txt +++ /dev/null @@ -1,114 +0,0 @@ -===================================== -Storing data persistently with Amazon -===================================== - -:Author: Rosangela Canino-Koning -:Date: June 7, 2010 - -If you want to save your data across instances -- that is, if you want -to have persistent data -- Amazon can do that for you, too. You need -to use the Amazon Elastic Block Storage service, which creates a virtual -hard drive that you can (virtually) attach to your EC2 instance. - -Note that EBS charges you based partly on how much disk space you have -allocated per month, rather than on connectivity or anything else, so even if -you don't have an EC2 machine running, you'll be paying Amazon for data -storage. - -You can read more about EBS `here `__. - -Prerequisites -------------- - -This tutorial assumes you've already set up an account on Amazon Web -Services, and that you've completed the EC2 tutorial to set up an -Amazon instance. - -Ask Amazon to create a new Elastic Block Storage Volume for you ---------------------------------------------------------------- - -At the AWS Management Console, on the left menu bar, click "Volumes". - -.. image:: images/ClickVolumes.png - :width: 90% - - -Click "Create Volume". - -.. image:: images/CreateVolume.png - :width: 90% - -Enter the desired size, and select the zone in which your instance is -running. The volume and instance must be in the same zone. Otherwise, -the volume cannot be attached to your instance. - -Then click "Create". - -.. image:: images/CreateVolumeWindow.png - :width: 90% - - -Wait for your volume to finish being created, then click "Attach Volume". - -.. image:: images/AttachVolume.png - :width: 90% - -Select the desired running instance. It will ask you for a device name -to attach; this should be '/dev/sdf', '/dev/sdg', etc. You can name it -anything up to at least 'i' or 'j'. Remember this for later -- it's -how the computer will know which disk to store data on! - -Click "Attach". - -.. image:: images/AttachVolumeWindow.png - :width: 90% - -When attachment is complete, connect to your instances via SSH. - -If the volume is newly created, you must format the volume. -**WARNING: ONLY DO THIS ONCE, WHEN YOU FIRST CREATE THE VOLUME. OTHERWISE, YOU WILL LOSE ALL YOUR -DATA**. -:: - - mkfs -t ext2 /dev/xvdf - -(If you used 'sdg' above, make it 'xvdg' etc. I know it's confusing.) - -It will ask you if you want to use the entire device -- say "y" for "yes. - -Then, mount the volume. You'll do this every time you attach the volume to an instance:: - - mkdir /data - mount /dev/xvdf /data - -Your drive is now ready to use -- it will be available under /data. -Files copied into that directory or directories underneath it will -be stored on your EBS volume. - -Shutting down your instance ---------------------------- - -Any volumes you have attached will automatically detach when you shut -down the instance. You can also stop all processes that are using the -volume, change out of the directory, and type :: - - cd - umount /data - -and then detach the volume via the AWS Web site. - -Snapshotting your volume ------------------------- - -Snapshots are backups of your volume that you can share with other people. -Snapshots are much more reliable long-term than volumes are, and you can use -them as a basis for creating a new volume (in which case the new volume will -start out containing all the data in the snapshot). So, if you upload some -raw data and want to work with it over a few weeks, we suggest: - - - create a volume and load the data onto the volume - - snapshot the original volume - - make a new volume from the snapshot, and delete the original volume - -You'll only be charged money for storing the snapshot and any *differences* -from the snapshot on the volumes based on that snapshot. diff --git a/doc/mrnaseq/using-screen.txt b/doc/mrnaseq/using-screen.txt deleted file mode 100644 index aa45c06..0000000 --- a/doc/mrnaseq/using-screen.txt +++ /dev/null @@ -1,58 +0,0 @@ -============== -Using 'screen' -============== - -:Author: Rosangela Canino-Koning -:Date: June 9, 2011 -:Last Updated: July 24, 2013 - -Persistent Sessions -------------------- - -Screen is a window manager for terminal sessions. Screen allows you to -run a terminal session, and then disconnect from the computer, and be -able to return to the session at a later date. - -To start screen, you run the screen command with a few options:: - - screen -S - -Where *sessionname* is any meaningful or descriptive title for your screen -session. This creates an independent terminal session, and connects you to it. - -Most commands within screen are composed of a prefix key-stroke, -followed by a command character. By default, the prefix is Ctrl-A. In -this tutorial Ctrl-A will represented by "C-a". - -Let's try a few screen commands. - -To disconnect from the session (while leaving it running!), type:: - - C-a d - -This session will remain active until you choose to end it, or you -reboot the computer. You can at this point safely disconnect from SSH, -and the screen session will continue to run. - -To reconnect to the session, make sure you're logged into the UNIX machine, -and type:: - - screen -r - -To illustrate managing multiple screen session, disconnect from the current -session, and create a new session with a second name.:: - - C-a d - screen -S - -Disconnect from the second session, and then list the available sessions:: - - C-a d - screen -list - -Note, typing *screen -r* with multiple active screen sessions will display -the same information. - -To reconnect to the first session, include its name after the -r.:: - - screen -r From 52b35867d0d2b18a464071c31488cff962c64612 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Mon, 23 Sep 2013 22:50:54 -0400 Subject: [PATCH 09/14] updated --- doc/2013-caltech-cemi.txt | 10 ++++ doc/2013-msu-zero-entry.txt | 10 ++++ doc/diginorm-2012/pipeline-notes.txt | 76 ++++++++++++++-------------- doc/index.txt | 4 ++ 4 files changed, 63 insertions(+), 37 deletions(-) create mode 100644 doc/2013-caltech-cemi.txt create mode 100644 doc/2013-msu-zero-entry.txt diff --git a/doc/2013-caltech-cemi.txt b/doc/2013-caltech-cemi.txt new file mode 100644 index 0000000..c5c3f66 --- /dev/null +++ b/doc/2013-caltech-cemi.txt @@ -0,0 +1,10 @@ +2013 Caltech workshop: Center for Environmental Microbial Interactions +====================================================================== + +Titus Brown and Chris Welcher ran this workshop from Sep 17, 2013 to +Sep 20, 2013 at Caltech. The workshop was focused on microbial data sets. + +The materials are available here: + + http://2013-caltech-workshop.readthedocs.org + diff --git a/doc/2013-msu-zero-entry.txt b/doc/2013-msu-zero-entry.txt new file mode 100644 index 0000000..6fa16ac --- /dev/null +++ b/doc/2013-msu-zero-entry.txt @@ -0,0 +1,10 @@ +2013 MSU "Zero-Entry" workshop: Computational Science for Biologists +==================================================================== + +Titus Brown and Tracy Teal ran this workshop on Sep 23 and Sep 25, 2013, +at Michigan State University. The workshop was focused on an introduction +to computational science, the shell, the cloud, and IPython Notebook. + +The materials are here:: + + http://2013-msu-zero-entry.readthedocs.org diff --git a/doc/diginorm-2012/pipeline-notes.txt b/doc/diginorm-2012/pipeline-notes.txt index f05a455..53be326 100644 --- a/doc/diginorm-2012/pipeline-notes.txt +++ b/doc/diginorm-2012/pipeline-notes.txt @@ -26,7 +26,7 @@ Starting up a machine and installing software First, start up an EC2 instance using starcluster:: - % starcluster start -o -s 1 -i m2.xlarge -n ami-999d49f0 pipeline + starcluster start -o -s 1 -i m2.xlarge -n ami-999d49f0 pipeline You can also do this via the AWS console; just use ami-999d49f0, and start an instance with 16gb or more of memory. @@ -37,50 +37,52 @@ notebook. Now, log in! :: - % starcluster sshmaster pipeline + starcluster sshmaster pipeline (or just ssh in however you would normally do it.) Once you're logged in, you'll need to install both 'screed' and 'khmer'. In this case we're going to use the versions tagged for the paper sub.:: - % cd /usr/local/src + cd /usr/local/share - % git clone git://github.com/ged-lab/screed.git - % cd screed - % git checkout 2012-paper-diginorm - % python setup.py install - % cd .. + git clone git://github.com/ged-lab/screed.git + cd screed + git checkout 2012-paper-diginorm + python setup.py install + cd .. - % git clone git://github.com/ged-lab/khmer.git - % cd khmer - % git checkout 2012-paper-diginorm - % make test - % cd .. + git clone git://github.com/ged-lab/khmer.git + cd khmer + git checkout 2012-paper-diginorm + make test + cd .. - % echo export PYTHONPATH=/usr/local/src/khmer/python >> ~/.bashrc - % echo 'export PATH=$PATH:/usr/local/src/khmer/scripts' >> ~/.bashrc - % echo 'export PATH=$PATH:/usr/local/src/khmer/sandbox' >> ~/.bashrc - % source ~/.bashrc + echo export PYTHONPATH=/usr/local/share/khmer/python >> ~/.bashrc + echo 'export PATH=$PATH:/usr/local/share/khmer/scripts' >> ~/.bashrc + echo 'export PATH=$PATH:/usr/local/share/khmer/sandbox' >> ~/.bashrc + source ~/.bashrc OK, now that these are both built, let's install two other things: the latest version of ipython notebook (you need 0.13dev, or later):: - % git clone https://github.com/ipython/ipython.git - % cd ipython - % python setup.py install + git clone https://github.com/ipython/ipython.git + cd ipython + python setup.py install + + pip install -U pyzmq and bowtie:: - % cd /mnt + cd /mnt - % curl -L -O http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip/download - % unzip download - % cp bowtie-0.12.7/bowtie{,-build} /usr/local/bin + curl -L -O http://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.7/bowtie-0.12.7-linux-x86_64.zip/download + unzip download + cp bowtie-0.12.7/bowtie{,-build} /usr/local/bin Finally, upgrade the latex install with a few recommended packages:: - % apt-get install texlive-latex-recommended + apt-get install -y texlive-latex-recommended OK, now all your software is installed, hurrah! @@ -90,26 +92,26 @@ Running the pipeline First, check out the source repository and grab the (...large) initial data sets:: - % git clone git://github.com/ged-lab/2012-paper-diginorm.git - % cd 2012-paper-diginorm + git clone https://github.com/ged-lab/2012-paper-diginorm.git + cd 2012-paper-diginorm - % curl -O https://s3.amazonaws.com/public.ged.msu.edu/2012-paper-diginorm/pipeline-data-new.tar.gz - % tar xzf pipeline-data-new.tar.gz + curl -O https://s3.amazonaws.com/public.ged.msu.edu/2012-paper-diginorm/pipeline-data-new.tar.gz + tar xzf pipeline-data-new.tar.gz Now go into the pipeline directory and run the pipeline. This will take 4-8 hours, so you might want to do it in 'screen' (see :doc:`../tutorials-2011/unix_long_jobs`). :: - % cd pipeline - % make KHMER=/usr/local/src/khmer + cd pipeline + make KHMER=/usr/local/share/khmer Once it successfully completes, copy the data over to the ../data/ directory:: - % make copydata + make copydata Run the ipython notebook server:: - % cd ../notebook - % ipython notebook --pylab=inline --no-browser --ip=* --port=80 & + cd ../notebook + ipython notebook --pylab=inline --no-browser --ip=* --port=80 & Connect into the ipython notebook (it will be running at 'http://'); if the above command succeeded but you can't connect in, you probably forgot to enable port 80 on your EC2 firewall. @@ -121,9 +123,9 @@ and select 'Run all'. Now go back to the command line and execute:: - % mv *.pdf ../ - % cd ../ - % make + mv *.pdf ../ + cd ../ + make and voila, 'diginorm.pdf' will contain the paper with the figures you just created. diff --git a/doc/index.txt b/doc/index.txt index e294ea5..20cf951 100644 --- a/doc/index.txt +++ b/doc/index.txt @@ -32,6 +32,10 @@ Contents: bioinformatics-courses + 2013-msu-zero-entry + + 2013-caltech-cemi + tutorials-2013/index 2013-hmp-assembly-webinar/index From 27b3a1ef92efde08b6494a3d5d178589083f389c Mon Sep 17 00:00:00 2001 From: Luiz Irber Date: Fri, 17 Jan 2014 11:49:08 -0500 Subject: [PATCH 10/14] Fix instruction for download Instructions referred curl, but the command used was wget. Fix correct flag (should be -o instead of -O). --- doc/tutorials-2013/bwa-tutorial.txt | 10 +++++----- 1 file changed, 5 insertions(+), 5 deletions(-) diff --git a/doc/tutorials-2013/bwa-tutorial.txt b/doc/tutorials-2013/bwa-tutorial.txt index 4ac49c2..01c8fc1 100644 --- a/doc/tutorials-2013/bwa-tutorial.txt +++ b/doc/tutorials-2013/bwa-tutorial.txt @@ -86,13 +86,13 @@ To actually do the mapping, we need to download and install bwa. First we are going to grab the source files for bwa from sourceforge, using curl. It is important to know that we need to specify a few -flags to let the program know that we want to keep the name (and -filetype) for the file (-O) and that curl should follow relative -hyperlinks (-L), to deal with redirection of the file site (or else -curl won't work with sourceforge).:: +flags to let the program know that we want to save the output to a +file (-o) instead of the default (print it on the screen) and that +curl should follow relative hyperlinks (-L), to deal with redirection +of the file site (or else curl won't work with sourceforge).:: cd /mnt - wget -O bwa-0.7.5.tar.bz2 http://sourceforge.net/projects/bio-bwa/files/bwa-0.7.5a.tar.bz2/download + curl -L -o bwa-0.7.5.tar.bz2 http://sourceforge.net/projects/bio-bwa/files/bwa-0.7.5a.tar.bz2/download Now we want to uncompress the tarball file using "tar". x extracts, v is verbose (telling you what it is doing), f skips prompting for each From f9b0bf4bf2023ac65a7f767330798f76d4751596 Mon Sep 17 00:00:00 2001 From: Michael Crusoe Date: Tue, 4 Feb 2014 15:59:05 -0500 Subject: [PATCH 11/14] add warnings about content not being maintained --- doc/2012-oslo-metagenomics/a-fake-metagenome.txt | 6 ++++++ doc/2012-oslo-metagenomics/a-real-genome.txt | 6 ++++++ doc/2012-oslo-metagenomics/index.txt | 6 ++++++ doc/2012-oslo-metagenomics/installing.txt | 7 +++++++ doc/2012-oslo-metagenomics/preparing-data.txt | 6 ++++++ doc/2013-04-assembly-workshop/assembly-with-a5.txt | 6 ++++++ doc/2013-04-assembly-workshop/assembly-with-velvet.txt | 6 ++++++ doc/2013-04-assembly-workshop/getting-data.txt | 6 ++++++ doc/2013-04-assembly-workshop/index.txt | 6 ++++++ doc/2013-04-assembly-workshop/installing-software.txt | 6 ++++++ doc/2013-04-assembly-workshop/mapping-reads.txt | 6 ++++++ doc/2013-04-assembly-workshop/running-blast.txt | 6 ++++++ .../trimming-and-quality-evaluation.txt | 6 ++++++ doc/2013-hmp-assembly-webinar/exploring-stamps-data.txt | 6 ++++++ doc/2013-hmp-assembly-webinar/index.txt | 6 ++++++ doc/2013-hmp-assembly-webinar/installing-assemblers.txt | 6 ++++++ doc/2013-hmp-assembly-webinar/playing-with-assemblers.txt | 6 ++++++ .../working-with-real-volumes-of-data.txt | 6 ++++++ doc/2013-stamps/1-quality.txt | 6 ++++++ doc/2013-stamps/1-setup.txt | 6 ++++++ doc/2013-stamps/2-quality.txt | 6 ++++++ doc/2013-stamps/3-diginorm.txt | 6 ++++++ doc/2013-stamps/4-partition.txt | 6 ++++++ doc/2013-stamps/5-assemble.txt | 6 ++++++ doc/2013-stamps/6-mapping-and-quantitation.txt | 6 ++++++ doc/2013-stamps/index.txt | 7 +++++-- doc/assembly-workshop/bowtie-mapping.txt | 6 ++++++ doc/aws-cheat-sheet.txt | 6 ++++++ doc/beacon-2011/week14-hw.txt | 6 ++++++ doc/beacon-2012/week7-khmer.txt | 6 ++++++ doc/beacon-2012/week7.txt | 6 ++++++ doc/fhl-course-exercise.txt | 6 ++++++ doc/hmp-workshop-adina/mock-pipeline.txt | 6 ++++++ doc/index.txt | 6 ++++++ doc/metag-assembly-2011/assembly-diff.txt | 6 ++++++ doc/metag-assembly-2011/diginorm.txt | 6 ++++++ doc/metag-assembly-2011/index.txt | 6 ++++++ doc/metag-assembly-2011/installing-khmer.txt | 6 ++++++ doc/metag-assembly-2011/khmer-scripts.txt | 6 ++++++ doc/metag-assembly-2011/partitioning.txt | 6 ++++++ doc/metag-assembly-2011/running-trinity.txt | 6 ++++++ doc/metag-assembly-2011/short-read-assembly-idba.txt | 6 ++++++ doc/metag-assembly-2011/short-read-assembly-metavelvet.txt | 6 ++++++ doc/metag-assembly-2011/short-read-assembly-velvet.txt | 6 ++++++ doc/metag-assembly-2011/starting-your-cloud-system.txt | 6 ++++++ doc/metag-assembly-2011/velvet-multik.txt | 6 ++++++ doc/nih-hmp-2012/index.txt | 6 ++++++ doc/stamps-2012/basic-partitioning-and-diginorm.txt | 6 ++++++ doc/stamps-2012/hmp-mock-diginorm.txt | 6 ++++++ doc/stamps-2012/index.txt | 6 ++++++ doc/stamps-2012/kmer-abundance-dists.txt | 6 ++++++ doc/stamps-2012/stamps-assembly-1.txt | 6 ++++++ doc/tutorials-2011/index.txt | 6 ++++++ doc/tutorials-2011/intro-scripting.txt | 6 ++++++ doc/tutorials-2011/mrnaseq-combined.txt | 6 ++++++ doc/tutorials-2011/short-read-assembly-velvet.txt | 6 ++++++ doc/tutorials-2012/day4.txt | 6 ++++++ doc/tutorials-2012/index.txt | 6 ++++++ doc/tutorials-2012/long-running-processes-and-assembly.txt | 6 ++++++ doc/tutorials-2013/assembling-ecoli-with-velvet.txt | 6 ++++++ doc/tutorials-2013/index.txt | 6 ++++++ doc/tutorials-2013/kmer-abundance-and-diginorm.txt | 6 ++++++ doc/tutorials-2013/short-read-quality-evaluation.txt | 6 ++++++ doc/tutorials/more-advanced-python-scripting.txt | 6 ++++++ doc/tutorials/short-read-assembly.txt | 6 ++++++ 65 files changed, 390 insertions(+), 2 deletions(-) diff --git a/doc/2012-oslo-metagenomics/a-fake-metagenome.txt b/doc/2012-oslo-metagenomics/a-fake-metagenome.txt index 43fce19..b8b6c49 100644 --- a/doc/2012-oslo-metagenomics/a-fake-metagenome.txt +++ b/doc/2012-oslo-metagenomics/a-fake-metagenome.txt @@ -1,6 +1,12 @@ A fake metagenome ================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Let's start by playing with a little fake metagenome. :: %% mkdir ~/metag/ diff --git a/doc/2012-oslo-metagenomics/a-real-genome.txt b/doc/2012-oslo-metagenomics/a-real-genome.txt index 2663e8f..d99019f 100644 --- a/doc/2012-oslo-metagenomics/a-real-genome.txt +++ b/doc/2012-oslo-metagenomics/a-real-genome.txt @@ -1,6 +1,12 @@ Assembling a real genome ======================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Normalize to 20:: %% cd ~/metag diff --git a/doc/2012-oslo-metagenomics/index.txt b/doc/2012-oslo-metagenomics/index.txt index 2d301b1..d787f85 100644 --- a/doc/2012-oslo-metagenomics/index.txt +++ b/doc/2012-oslo-metagenomics/index.txt @@ -2,6 +2,12 @@ A Short Metagenomics Workshop in Oslo (Dec 2012) ================================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Hipchat room: https://www.hipchat.com/gSoSjn5lP Other links: diff --git a/doc/2012-oslo-metagenomics/installing.txt b/doc/2012-oslo-metagenomics/installing.txt index 2018e03..cf32fe4 100644 --- a/doc/2012-oslo-metagenomics/installing.txt +++ b/doc/2012-oslo-metagenomics/installing.txt @@ -1,6 +1,13 @@ Installing the necessary software on the cod servers ==================================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + + This should all run on cod3:: %% ssh guestXXX@cod3.uio.no diff --git a/doc/2012-oslo-metagenomics/preparing-data.txt b/doc/2012-oslo-metagenomics/preparing-data.txt index 1f0bf30..9abaf25 100644 --- a/doc/2012-oslo-metagenomics/preparing-data.txt +++ b/doc/2012-oslo-metagenomics/preparing-data.txt @@ -2,6 +2,12 @@ Preparing data from raw ======================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + I grabbed the PhiX, DH10B, and MG1655 data sets from `Illumina's public data repo `__. diff --git a/doc/2013-04-assembly-workshop/assembly-with-a5.txt b/doc/2013-04-assembly-workshop/assembly-with-a5.txt index 5f34223..f046787 100644 --- a/doc/2013-04-assembly-workshop/assembly-with-a5.txt +++ b/doc/2013-04-assembly-workshop/assembly-with-a5.txt @@ -1,6 +1,12 @@ Assembling microbial genomes with A5 ==================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Let's try using a different assembly pipeline -- the A5 pipeline, from `An Integrated Pipeline for de Novo Assembly of Microbial Genomes `__. diff --git a/doc/2013-04-assembly-workshop/assembly-with-velvet.txt b/doc/2013-04-assembly-workshop/assembly-with-velvet.txt index 3e87b50..c7e3877 100644 --- a/doc/2013-04-assembly-workshop/assembly-with-velvet.txt +++ b/doc/2013-04-assembly-workshop/assembly-with-velvet.txt @@ -1,6 +1,12 @@ Assembling sequences with Velvet ================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Start at the EC2 command line prompt -- :: cd /mnt diff --git a/doc/2013-04-assembly-workshop/getting-data.txt b/doc/2013-04-assembly-workshop/getting-data.txt index 04750f2..13ac6f1 100644 --- a/doc/2013-04-assembly-workshop/getting-data.txt +++ b/doc/2013-04-assembly-workshop/getting-data.txt @@ -1,6 +1,12 @@ Getting the data ================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Start at your EC2 prompt, then type :: cd /mnt diff --git a/doc/2013-04-assembly-workshop/index.txt b/doc/2013-04-assembly-workshop/index.txt index 25808ab..13e8a7f 100644 --- a/doc/2013-04-assembly-workshop/index.txt +++ b/doc/2013-04-assembly-workshop/index.txt @@ -1,6 +1,12 @@ 2013 MSU Assembly Workshop ========================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Tracy Teal Titus Brown diff --git a/doc/2013-04-assembly-workshop/installing-software.txt b/doc/2013-04-assembly-workshop/installing-software.txt index c7c534e..4eee44e 100644 --- a/doc/2013-04-assembly-workshop/installing-software.txt +++ b/doc/2013-04-assembly-workshop/installing-software.txt @@ -1,6 +1,12 @@ Packages to install =================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + These command should work on the default `StarCluster `__ AMI, ami-999d49f0. These have already been pre-installed on the course AMI, ami-d6e581bf. diff --git a/doc/2013-04-assembly-workshop/mapping-reads.txt b/doc/2013-04-assembly-workshop/mapping-reads.txt index 2af8fc8..c1519a6 100644 --- a/doc/2013-04-assembly-workshop/mapping-reads.txt +++ b/doc/2013-04-assembly-workshop/mapping-reads.txt @@ -1,6 +1,12 @@ Mapping reads to a reference ---------------------------- +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Create a new directory to do the mapping in:: cd /mnt diff --git a/doc/2013-04-assembly-workshop/running-blast.txt b/doc/2013-04-assembly-workshop/running-blast.txt index d06a310..128802f 100644 --- a/doc/2013-04-assembly-workshop/running-blast.txt +++ b/doc/2013-04-assembly-workshop/running-blast.txt @@ -1,6 +1,12 @@ Installing and running BLAST ============================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Everyone wants to BLAST! How do we do it? First, install NCBI BLAST:: diff --git a/doc/2013-04-assembly-workshop/trimming-and-quality-evaluation.txt b/doc/2013-04-assembly-workshop/trimming-and-quality-evaluation.txt index 12bfd1e..7154a7a 100644 --- a/doc/2013-04-assembly-workshop/trimming-and-quality-evaluation.txt +++ b/doc/2013-04-assembly-workshop/trimming-and-quality-evaluation.txt @@ -1,6 +1,12 @@ Trimming and quality evaluation of your sequences ================================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Start at the EC2 login prompt. Then, :: cd /mnt diff --git a/doc/2013-hmp-assembly-webinar/exploring-stamps-data.txt b/doc/2013-hmp-assembly-webinar/exploring-stamps-data.txt index 4b089ef..961c355 100644 --- a/doc/2013-hmp-assembly-webinar/exploring-stamps-data.txt +++ b/doc/2013-hmp-assembly-webinar/exploring-stamps-data.txt @@ -1,6 +1,12 @@ Exploring a simple simulated data set ===================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Let's take a look at some k-mer abundance distributions, and the effects of various activities on them. diff --git a/doc/2013-hmp-assembly-webinar/index.txt b/doc/2013-hmp-assembly-webinar/index.txt index f1ce47f..d9cb61c 100644 --- a/doc/2013-hmp-assembly-webinar/index.txt +++ b/doc/2013-hmp-assembly-webinar/index.txt @@ -1,6 +1,12 @@ 2013 HMP Assembly Webinar -- electronic materials ================================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :author: Titus Brown :date: May 6th, 2013 diff --git a/doc/2013-hmp-assembly-webinar/installing-assemblers.txt b/doc/2013-hmp-assembly-webinar/installing-assemblers.txt index f4bbed7..da40df1 100644 --- a/doc/2013-hmp-assembly-webinar/installing-assemblers.txt +++ b/doc/2013-hmp-assembly-webinar/installing-assemblers.txt @@ -1,6 +1,12 @@ Installing assemblers (and other necessary software) ==================================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + The first thing we need to do to make use of our Amazon machine is to install a bunch of assemblers. diff --git a/doc/2013-hmp-assembly-webinar/playing-with-assemblers.txt b/doc/2013-hmp-assembly-webinar/playing-with-assemblers.txt index 116e55d..d708254 100644 --- a/doc/2013-hmp-assembly-webinar/playing-with-assemblers.txt +++ b/doc/2013-hmp-assembly-webinar/playing-with-assemblers.txt @@ -1,6 +1,12 @@ Playing with metagenome assemblers ================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :author: C. Titus Brown :date: May 7, 2013 diff --git a/doc/2013-hmp-assembly-webinar/working-with-real-volumes-of-data.txt b/doc/2013-hmp-assembly-webinar/working-with-real-volumes-of-data.txt index b378899..fec0b82 100644 --- a/doc/2013-hmp-assembly-webinar/working-with-real-volumes-of-data.txt +++ b/doc/2013-hmp-assembly-webinar/working-with-real-volumes-of-data.txt @@ -1,6 +1,12 @@ Preparing the data you just used - filtering, normalizing, partitiong ===================================================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :author: C. Titus Brown :date: May 7, 2013 diff --git a/doc/2013-stamps/1-quality.txt b/doc/2013-stamps/1-quality.txt index 91f1d84..b0415b6 100644 --- a/doc/2013-stamps/1-quality.txt +++ b/doc/2013-stamps/1-quality.txt @@ -2,6 +2,12 @@ 1. Adapter trimming and quality filtering ========================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Configure your environment:: echo 'export PYTHONPATH=$PYTHONPATH:/class/stamps-software/share/khmer/python' >> ~/.bash_login diff --git a/doc/2013-stamps/1-setup.txt b/doc/2013-stamps/1-setup.txt index 9fd5434..fc545f2 100644 --- a/doc/2013-stamps/1-setup.txt +++ b/doc/2013-stamps/1-setup.txt @@ -2,6 +2,12 @@ 1. Setup ======== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Configure your environment:: echo 'export PYTHONPATH=$PYTHONPATH:/class/stamps-software/share/khmer/python' >> ~/.bash_login diff --git a/doc/2013-stamps/2-quality.txt b/doc/2013-stamps/2-quality.txt index b258e97..cfa1319 100644 --- a/doc/2013-stamps/2-quality.txt +++ b/doc/2013-stamps/2-quality.txt @@ -2,6 +2,12 @@ 2. Adapter trimming and quality filtering ========================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + .. note:: Activate your virtual environment and make sure you're running in screen! (see :doc:`../mrnaseq/using-screen`) diff --git a/doc/2013-stamps/3-diginorm.txt b/doc/2013-stamps/3-diginorm.txt index 407d8e6..0884ea1 100644 --- a/doc/2013-stamps/3-diginorm.txt +++ b/doc/2013-stamps/3-diginorm.txt @@ -2,6 +2,12 @@ 3. Running digital normalization ================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + .. note:: Activate your virtual environment and make sure you're running in screen! diff --git a/doc/2013-stamps/4-partition.txt b/doc/2013-stamps/4-partition.txt index adbf5d7..7006e24 100644 --- a/doc/2013-stamps/4-partition.txt +++ b/doc/2013-stamps/4-partition.txt @@ -2,6 +2,12 @@ 4. Partitioning =============== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + .. note:: Activate your virtual environment and make sure you're running in screen! diff --git a/doc/2013-stamps/5-assemble.txt b/doc/2013-stamps/5-assemble.txt index aec7b7a..342a332 100644 --- a/doc/2013-stamps/5-assemble.txt +++ b/doc/2013-stamps/5-assemble.txt @@ -2,6 +2,12 @@ 5. Assembling ============= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + At last! All that filtering and diginorming is done, and we can get down to the serious business of assembling. Huzzah! diff --git a/doc/2013-stamps/6-mapping-and-quantitation.txt b/doc/2013-stamps/6-mapping-and-quantitation.txt index d786889..7b0863a 100644 --- a/doc/2013-stamps/6-mapping-and-quantitation.txt +++ b/doc/2013-stamps/6-mapping-and-quantitation.txt @@ -2,6 +2,12 @@ 6. Mapping and abundance quantitation ===================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Let's do some simple mapping to do abundance estimation in the group 5 assembly. diff --git a/doc/2013-stamps/index.txt b/doc/2013-stamps/index.txt index f47aceb..3bd1246 100644 --- a/doc/2013-stamps/index.txt +++ b/doc/2013-stamps/index.txt @@ -1,8 +1,11 @@ The STAMPS Metagenome Assembly Tutorial ======================================= -**Note:** This tutorial is being updated and maintained over at -`the khmer protocols `__. +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. .. toctree:: :maxdepth: 1 diff --git a/doc/assembly-workshop/bowtie-mapping.txt b/doc/assembly-workshop/bowtie-mapping.txt index 0ff405c..45e4156 100644 --- a/doc/assembly-workshop/bowtie-mapping.txt +++ b/doc/assembly-workshop/bowtie-mapping.txt @@ -2,6 +2,12 @@ Mapping with bowtie =================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + bowtie is an open source mapping program that's fast and easy to use. Installing bowtie diff --git a/doc/aws-cheat-sheet.txt b/doc/aws-cheat-sheet.txt index d61427f..d98c4d2 100644 --- a/doc/aws-cheat-sheet.txt +++ b/doc/aws-cheat-sheet.txt @@ -2,6 +2,12 @@ Amazon Web Services cheat sheet ------------------------------- +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + See :doc:`tutorials/renting-a-computer-from-amazon` for a tutorial on starting up an Amazon EC2 system. diff --git a/doc/beacon-2011/week14-hw.txt b/doc/beacon-2011/week14-hw.txt index 6f34798..b5151af 100644 --- a/doc/beacon-2011/week14-hw.txt +++ b/doc/beacon-2011/week14-hw.txt @@ -48,6 +48,12 @@ the evolution techniques? Computational HW (due Wednesday, Dec 7th, at midnight) ------------------------------------------------------ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Run through :doc:`../tutorials-2011/short-read-assembly-velvet`, using AMI ami-67fe300e. The data is on snapshot snap-000d346e, so you'll need to create a volume from that snapshot and mount it as /dev/xvdf. (See diff --git a/doc/beacon-2012/week7-khmer.txt b/doc/beacon-2012/week7-khmer.txt index 1e464d6..2af9ab3 100644 --- a/doc/beacon-2012/week7-khmer.txt +++ b/doc/beacon-2012/week7-khmer.txt @@ -1,6 +1,12 @@ Installing khmer. ================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + So far, we've mostly been working with pre-installed software -- which just means that someone *else* has gone to the trouble of finding it, downloading it, compiling it, and installing it. Velvet was your first diff --git a/doc/beacon-2012/week7.txt b/doc/beacon-2012/week7.txt index 85f5bd1..622b5b5 100644 --- a/doc/beacon-2012/week7.txt +++ b/doc/beacon-2012/week7.txt @@ -1,6 +1,12 @@ Week 7 - More assembly; k-mers ============================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Reminder: a k-mer is just a fixed length word of DNA. Re-read the Miller paper sections on de Bruijn graphs if you need reminding! diff --git a/doc/fhl-course-exercise.txt b/doc/fhl-course-exercise.txt index 9b21fb9..93e2305 100644 --- a/doc/fhl-course-exercise.txt +++ b/doc/fhl-course-exercise.txt @@ -2,6 +2,12 @@ FHL Course Exercise: mRNAseq expression analysis ================================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + In this exercise, we're going to: - rent a computer from Amazon diff --git a/doc/hmp-workshop-adina/mock-pipeline.txt b/doc/hmp-workshop-adina/mock-pipeline.txt index 52b38d4..5e68070 100644 --- a/doc/hmp-workshop-adina/mock-pipeline.txt +++ b/doc/hmp-workshop-adina/mock-pipeline.txt @@ -1,6 +1,12 @@ From sequence to assembly, HMP mock community example ====================================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + We're going to demonstrate the pipeline we use to tackle complex metagenomic datasets. Our goal is to reduce to the dataset size to be smaller, disconnected partitions which one could then use for whatever needs you might have. In this example, we demonstrate the assembly and annotation of a metagenome using these methods. To summarize, the process includes 1) digital normalization to a specified coverage of quality-trimmed sequencing reads, 2) partitioning of disconnected subsets of reads, 3) removal of sequencing artifacts from the largest subset, 4) partitioning of the artifact-removed subset, 5) assembly of the partitions, 6) annotation of partitions with MG-RAST, and 7) recovering abundance information from raw reads for annotated ORFs. diff --git a/doc/index.txt b/doc/index.txt index e294ea5..5f9f670 100644 --- a/doc/index.txt +++ b/doc/index.txt @@ -6,6 +6,12 @@ Welcome to ANGUS! ================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + ANGUS is a site built around the `2010 course on Analyzing Next-Generation Sequencing Data `__. It diff --git a/doc/metag-assembly-2011/assembly-diff.txt b/doc/metag-assembly-2011/assembly-diff.txt index 67ba5fa..28d4684 100644 --- a/doc/metag-assembly-2011/assembly-diff.txt +++ b/doc/metag-assembly-2011/assembly-diff.txt @@ -1,6 +1,12 @@ Comparing assemblies done with different k values ================================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Here we're going to answer the age-old question: which is better, k=31 or k=33? diff --git a/doc/metag-assembly-2011/diginorm.txt b/doc/metag-assembly-2011/diginorm.txt index 22399d3..34ad7dc 100644 --- a/doc/metag-assembly-2011/diginorm.txt +++ b/doc/metag-assembly-2011/diginorm.txt @@ -1,6 +1,12 @@ Digital normalization ===================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + One approach to dealing with big data sets is to throw out redundant reads, which is pretty much what digital normalization does. diff --git a/doc/metag-assembly-2011/index.txt b/doc/metag-assembly-2011/index.txt index bb5a7b8..5b4e782 100644 --- a/doc/metag-assembly-2011/index.txt +++ b/doc/metag-assembly-2011/index.txt @@ -1,6 +1,12 @@ December 2011 / Metagenome Assembly workshop ============================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Contents: .. toctree:: diff --git a/doc/metag-assembly-2011/installing-khmer.txt b/doc/metag-assembly-2011/installing-khmer.txt index 8300192..3134257 100644 --- a/doc/metag-assembly-2011/installing-khmer.txt +++ b/doc/metag-assembly-2011/installing-khmer.txt @@ -1,6 +1,12 @@ Installing khmer ================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + khmer is a toolkit that we have been building here at MSU. It contains a bunch of scripts and libraries that are of varying utility. Below, we'll install it on our EC2 machine for later use. diff --git a/doc/metag-assembly-2011/khmer-scripts.txt b/doc/metag-assembly-2011/khmer-scripts.txt index 9d7642b..2bd9255 100644 --- a/doc/metag-assembly-2011/khmer-scripts.txt +++ b/doc/metag-assembly-2011/khmer-scripts.txt @@ -2,6 +2,12 @@ Useful Khmer Scripts ==================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :Author: Adina Howe :Date: December 11, 2011 :Last Updated: December 11, 2011 (Adina) diff --git a/doc/metag-assembly-2011/partitioning.txt b/doc/metag-assembly-2011/partitioning.txt index c78b8e1..a0f35a7 100644 --- a/doc/metag-assembly-2011/partitioning.txt +++ b/doc/metag-assembly-2011/partitioning.txt @@ -1,6 +1,12 @@ Partitioning ============ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + As part of our efforts to scale metagenome assembly, we've developed an IDBA/MetaVelvet-like approach that not only subdivides the reads into disconnected components but does so in less memory than either of the diff --git a/doc/metag-assembly-2011/running-trinity.txt b/doc/metag-assembly-2011/running-trinity.txt index 683d304..be65c67 100644 --- a/doc/metag-assembly-2011/running-trinity.txt +++ b/doc/metag-assembly-2011/running-trinity.txt @@ -1,6 +1,12 @@ Running Trinity in da cloud =========================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Follow :doc:`starting-your-cloud-system` up until "Getting data". Use the AMI (Amazon Machine Image) ami-2946a740 (Debian 6.0). diff --git a/doc/metag-assembly-2011/short-read-assembly-idba.txt b/doc/metag-assembly-2011/short-read-assembly-idba.txt index 3ef4236..427ffdf 100644 --- a/doc/metag-assembly-2011/short-read-assembly-idba.txt +++ b/doc/metag-assembly-2011/short-read-assembly-idba.txt @@ -2,6 +2,12 @@ Short Read Assembly with Meta-IDBA ================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :Author: Adina Howe :Date: December 11, 2011 :Last Updated: December 11, 2011 (Adina) diff --git a/doc/metag-assembly-2011/short-read-assembly-metavelvet.txt b/doc/metag-assembly-2011/short-read-assembly-metavelvet.txt index 615b668..284d0a5 100644 --- a/doc/metag-assembly-2011/short-read-assembly-metavelvet.txt +++ b/doc/metag-assembly-2011/short-read-assembly-metavelvet.txt @@ -2,6 +2,12 @@ Short Read Assembly with Meta-Velvet =============================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :Author: Adina Howe :Date: December 11, 2011 :Last Updated: December 11, 2011 (Adina) diff --git a/doc/metag-assembly-2011/short-read-assembly-velvet.txt b/doc/metag-assembly-2011/short-read-assembly-velvet.txt index e44183a..30657c1 100644 --- a/doc/metag-assembly-2011/short-read-assembly-velvet.txt +++ b/doc/metag-assembly-2011/short-read-assembly-velvet.txt @@ -2,6 +2,12 @@ Short Read Assembly with Velvet ================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :Author: Jason Pell, Adina Howe, Titus Brown :Date: June 7, 2011 :Last Updated: December 11, 2011 (adina) diff --git a/doc/metag-assembly-2011/starting-your-cloud-system.txt b/doc/metag-assembly-2011/starting-your-cloud-system.txt index d1994d5..7a29596 100644 --- a/doc/metag-assembly-2011/starting-your-cloud-system.txt +++ b/doc/metag-assembly-2011/starting-your-cloud-system.txt @@ -2,6 +2,12 @@ Starting your cloud system ========================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Introducing Amazon EC2 ---------------------- diff --git a/doc/metag-assembly-2011/velvet-multik.txt b/doc/metag-assembly-2011/velvet-multik.txt index 8af7ac7..1d720b7 100644 --- a/doc/metag-assembly-2011/velvet-multik.txt +++ b/doc/metag-assembly-2011/velvet-multik.txt @@ -1,6 +1,12 @@ Merging Velvet multi-k assemblies ================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Patrick Chain's group at LANL has developed an approach to merge regular Velvet assemblies with multiple ks using a program called minimus2. Let's try it out! diff --git a/doc/nih-hmp-2012/index.txt b/doc/nih-hmp-2012/index.txt index 4d3ed73..493126d 100644 --- a/doc/nih-hmp-2012/index.txt +++ b/doc/nih-hmp-2012/index.txt @@ -2,6 +2,12 @@ NIH HMP Workshop / Knight Lab - Apr 2012 ======================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Metagenomic assembly in the cloud ================================= diff --git a/doc/stamps-2012/basic-partitioning-and-diginorm.txt b/doc/stamps-2012/basic-partitioning-and-diginorm.txt index d988117..78c01f2 100644 --- a/doc/stamps-2012/basic-partitioning-and-diginorm.txt +++ b/doc/stamps-2012/basic-partitioning-and-diginorm.txt @@ -1,6 +1,12 @@ Basic partitioning and digital normalization ============================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + First, make sure you have activated bioware and installed khmer in your account; see :doc:`stamps-assembly-1` for more info. diff --git a/doc/stamps-2012/hmp-mock-diginorm.txt b/doc/stamps-2012/hmp-mock-diginorm.txt index c3ac9a7..ab18c21 100644 --- a/doc/stamps-2012/hmp-mock-diginorm.txt +++ b/doc/stamps-2012/hmp-mock-diginorm.txt @@ -1,6 +1,12 @@ Applying digital normalization and partitioning to real data ============================================================ +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Let's apply partitioning and digital normalization to real HMP data. (Hint: it works...) This will take 4-5 hours to run from start to finish. diff --git a/doc/stamps-2012/index.txt b/doc/stamps-2012/index.txt index d7250f5..a1b163f 100644 --- a/doc/stamps-2012/index.txt +++ b/doc/stamps-2012/index.txt @@ -2,6 +2,12 @@ Strategies and Techniques for Analyzing Microbial Population Structure (2012) ============================================================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + (This is just the metagenomics sequence assembly section of the course! Main course page: https://stamps.mbl.edu/index.php/Main_Page) diff --git a/doc/stamps-2012/kmer-abundance-dists.txt b/doc/stamps-2012/kmer-abundance-dists.txt index 9fa8c71..22ed2c5 100644 --- a/doc/stamps-2012/kmer-abundance-dists.txt +++ b/doc/stamps-2012/kmer-abundance-dists.txt @@ -1,6 +1,12 @@ Calculating k-mer abundance distributions ========================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Make sure you've got khmer installed (as in :doc:`stamps-assembly-1`) as well as the 'bioware' module loaded. Log into your server and create a new directory:: diff --git a/doc/stamps-2012/stamps-assembly-1.txt b/doc/stamps-2012/stamps-assembly-1.txt index f165c17..b76c430 100644 --- a/doc/stamps-2012/stamps-assembly-1.txt +++ b/doc/stamps-2012/stamps-assembly-1.txt @@ -1,6 +1,12 @@ Metagenomic assembly - STAMPS 2012, day 1 ========================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + C. Titus Brown **Note**: this is an adaptation of a 2011 set of tutorials, available diff --git a/doc/tutorials-2011/index.txt b/doc/tutorials-2011/index.txt index 9aa3d3e..62ca80a 100644 --- a/doc/tutorials-2011/index.txt +++ b/doc/tutorials-2011/index.txt @@ -2,6 +2,12 @@ NGS Workshop Tutorials (2011 version) ===================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + .. toctree:: :maxdepth: 2 diff --git a/doc/tutorials-2011/intro-scripting.txt b/doc/tutorials-2011/intro-scripting.txt index d049fac..da12781 100644 --- a/doc/tutorials-2011/intro-scripting.txt +++ b/doc/tutorials-2011/intro-scripting.txt @@ -140,6 +140,12 @@ the script into a file, or download it from https://raw.github.com/ctb/edda/mast Other Scripts ------------- +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + FASTQ to FASTA Converter: https://raw.github.com/ctb/khmer/master/scripts/fastq-to-fasta.py Strip and Split for Assembly: https://raw.github.com/ctb/khmer/master/scripts/strip-and-split-for-assembly.py Quality Trimming: https://raw.github.com/ctb/khmer/master/scripts/quality-trim.py diff --git a/doc/tutorials-2011/mrnaseq-combined.txt b/doc/tutorials-2011/mrnaseq-combined.txt index 75b35b0..c760d2d 100644 --- a/doc/tutorials-2011/mrnaseq-combined.txt +++ b/doc/tutorials-2011/mrnaseq-combined.txt @@ -2,6 +2,12 @@ A simple mRNAseq analysis pipeline ================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Start up an instance (ami-ea837b83). If you want to run your own assembly, you'll need at least 30 GB of RAM, so a high-memory double-extra large instance. If not, you only need the basic "large" diff --git a/doc/tutorials-2011/short-read-assembly-velvet.txt b/doc/tutorials-2011/short-read-assembly-velvet.txt index 71358c3..85ee1da 100644 --- a/doc/tutorials-2011/short-read-assembly-velvet.txt +++ b/doc/tutorials-2011/short-read-assembly-velvet.txt @@ -2,6 +2,12 @@ Short Read Assembly with Velvet =============================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :Author: Jason Pell :Date: June 7, 2011 :Last Updated: June 9, 2011 diff --git a/doc/tutorials-2012/day4.txt b/doc/tutorials-2012/day4.txt index df6b98d..0f40bec 100644 --- a/doc/tutorials-2012/day4.txt +++ b/doc/tutorials-2012/day4.txt @@ -2,6 +2,12 @@ Day 4: Assembly ============== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Thursday, June 7th, 2012 .. toctree:: diff --git a/doc/tutorials-2012/index.txt b/doc/tutorials-2012/index.txt index a4044fd..066eb72 100644 --- a/doc/tutorials-2012/index.txt +++ b/doc/tutorials-2012/index.txt @@ -2,6 +2,12 @@ Next-Gen Sequence Analysis Workshop (2012) ========================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Your AMI is **ami-00ca6a69** and the ipython notebook password is 'ngs'. .. toctree:: diff --git a/doc/tutorials-2012/long-running-processes-and-assembly.txt b/doc/tutorials-2012/long-running-processes-and-assembly.txt index d314292..181f9fc 100644 --- a/doc/tutorials-2012/long-running-processes-and-assembly.txt +++ b/doc/tutorials-2012/long-running-processes-and-assembly.txt @@ -1,6 +1,12 @@ Running processes (like assemblers) for a long time =================================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + In this tutorial, we'll do two things: first, we'll introduce a program called 'screen' that lets you run diff --git a/doc/tutorials-2013/assembling-ecoli-with-velvet.txt b/doc/tutorials-2013/assembling-ecoli-with-velvet.txt index 594f334..74efd30 100644 --- a/doc/tutorials-2013/assembling-ecoli-with-velvet.txt +++ b/doc/tutorials-2013/assembling-ecoli-with-velvet.txt @@ -2,6 +2,12 @@ Assembling E. coli sequences with Velvet ======================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Install screed:: cd /usr/local/share diff --git a/doc/tutorials-2013/index.txt b/doc/tutorials-2013/index.txt index 8ab23e3..cc5fef4 100644 --- a/doc/tutorials-2013/index.txt +++ b/doc/tutorials-2013/index.txt @@ -2,6 +2,12 @@ Next-Gen Sequence Analysis Workshop (2013) ========================================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + This is the schedule for the `2013 MSU NGS course `__, which ran from June 10th to June 20th, 2013. If you're interested in this course in 2014, please see `the 2014 announcement `__. =============== ============================================================= diff --git a/doc/tutorials-2013/kmer-abundance-and-diginorm.txt b/doc/tutorials-2013/kmer-abundance-and-diginorm.txt index 1e0a184..adacbe1 100644 --- a/doc/tutorials-2013/kmer-abundance-and-diginorm.txt +++ b/doc/tutorials-2013/kmer-abundance-and-diginorm.txt @@ -2,6 +2,12 @@ Calculating k-mer abundance distributions ========================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + Start up an m1.large. Install screed:: diff --git a/doc/tutorials-2013/short-read-quality-evaluation.txt b/doc/tutorials-2013/short-read-quality-evaluation.txt index 8f77eae..628a2ec 100644 --- a/doc/tutorials-2013/short-read-quality-evaluation.txt +++ b/doc/tutorials-2013/short-read-quality-evaluation.txt @@ -2,6 +2,12 @@ Evaluating the quality of your short reads, and trimming them ============================================================= +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + As useful as BLAST is, we really want to get into sequencing data, right? One of the first steps you must do with your data is evaluate its quality and throw away bad sequences. diff --git a/doc/tutorials/more-advanced-python-scripting.txt b/doc/tutorials/more-advanced-python-scripting.txt index 1d22f9b..f8184fe 100644 --- a/doc/tutorials/more-advanced-python-scripting.txt +++ b/doc/tutorials/more-advanced-python-scripting.txt @@ -103,6 +103,12 @@ For example... More FASTA stuff, with screed ----------------------------- +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + ...we can use the screed FASTA/FASTQ parser library to make FASTA and FASTQ files look like dictionaries. diff --git a/doc/tutorials/short-read-assembly.txt b/doc/tutorials/short-read-assembly.txt index edda436..2847033 100644 --- a/doc/tutorials/short-read-assembly.txt +++ b/doc/tutorials/short-read-assembly.txt @@ -2,6 +2,12 @@ Short Read Assembly =================== +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + :Author: Jason Pell :Date: June 5, 2010 :Last Updated: September 13, 2010 From e82f8a89cc46550c6f71f6c7609d864712fe7ac8 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Sun, 16 Feb 2014 21:26:14 -0500 Subject: [PATCH 12/14] added zero-entry links --- doc/2013-msu-zero-entry.txt | 7 ++++--- doc/index.txt | 2 ++ 2 files changed, 6 insertions(+), 3 deletions(-) diff --git a/doc/2013-msu-zero-entry.txt b/doc/2013-msu-zero-entry.txt index 6fa16ac..e83ade2 100644 --- a/doc/2013-msu-zero-entry.txt +++ b/doc/2013-msu-zero-entry.txt @@ -1,9 +1,10 @@ 2013 MSU "Zero-Entry" workshop: Computational Science for Biologists ==================================================================== -Titus Brown and Tracy Teal ran this workshop on Sep 23 and Sep 25, 2013, -at Michigan State University. The workshop was focused on an introduction -to computational science, the shell, the cloud, and IPython Notebook. +Titus Brown, Tracy Teal, and Jory Schossau ran this workshop on Sep 23 +and Sep 25, 2013, at Michigan State University. The workshop was +focused on an introduction to computational science, the shell, the +cloud, and IPython Notebook. The materials are here:: diff --git a/doc/index.txt b/doc/index.txt index 545a656..6507837 100644 --- a/doc/index.txt +++ b/doc/index.txt @@ -38,6 +38,8 @@ Contents: bioinformatics-courses + 2013-uw-zero-entry + 2013-msu-zero-entry 2013-caltech-cemi From 3f5e2a586dd831307a7e92b3a673759fb9f100e7 Mon Sep 17 00:00:00 2001 From: "C. Titus Brown" Date: Sun, 16 Feb 2014 21:32:09 -0500 Subject: [PATCH 13/14] changed index warning to a note --- doc/index.txt | 10 +++++----- 1 file changed, 5 insertions(+), 5 deletions(-) diff --git a/doc/index.txt b/doc/index.txt index 6507837..592c6c0 100644 --- a/doc/index.txt +++ b/doc/index.txt @@ -6,11 +6,11 @@ Welcome to ANGUS! ================= -.. Warning:: These documents are not maintained and their instructions may be - out of date. However the GED Lab does maintain the `khmer protocols - `__ which may cover similar - topics. See also the `installation instructions for the current version - of the khmer project `__. +.. note:: These tutorials are checked at the time of posting, but many + are now quite old and out of date. The GED Lab does maintain + a set of up-to-date tutorials on sequence analysis, the + `khmer protocols `__, + which may cover similar topics. ANGUS is a site built around the `2010 course on Analyzing Next-Generation Sequencing Data From 16fb46a9615cf665dc82004c45e2a0df32fc4474 Mon Sep 17 00:00:00 2001 From: "Michael R. Crusoe" Date: Mon, 3 Mar 2014 21:32:07 -0500 Subject: [PATCH 14/14] one more warning --- doc/diginorm-2012/tutorial.txt | 6 ++++++ 1 file changed, 6 insertions(+) diff --git a/doc/diginorm-2012/tutorial.txt b/doc/diginorm-2012/tutorial.txt index fd8bcc1..e6b2355 100644 --- a/doc/diginorm-2012/tutorial.txt +++ b/doc/diginorm-2012/tutorial.txt @@ -4,6 +4,12 @@ A tutorial in basic digital normalization :Date: March 25, 2012 +.. Warning:: These documents are not maintained and their instructions may be + out of date. However the GED Lab does maintain the `khmer protocols + `__ which may cover similar + topics. See also the `installation instructions for the current version + of the khmer project `__. + See http://arxiv.org/abs/1203.4802 for more information on digital normalization.