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cse527_project_report.lyx
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#LyX 2.0 created this file. For more info see http://www.lyx.org/
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\begin_body
\begin_layout Title
CSE527 Au12 Project Report -- A Genome Wide Motif Search
\end_layout
\begin_layout Author
James Youngquist <[email protected]>
\end_layout
\begin_layout Section
Introduction
\end_layout
\begin_layout Standard
This report describes a method for searching an entire genome for motifs.
While I limited my searches to nucleotide motifs on DNA regions outside
of genes, there is nothing in principle that prevents this method from
being used for codon searches within genes.
However, unless specified otherwise, all further references to searching,
motifs, etc.
refer to nucleotide motifs on non-gene regions.
The technique described here was independently developed and the author
is not aware of it being used elsewhere.
\end_layout
\begin_layout Standard
Motifs are repeating sequences that are found throughout a genome and which
may have biological significance in their own right.
For example, the TATA box is a motif found in the promoter regions of some
genes that acts as a binding site for transcription factors (TF) [1].
Because TF binding sites can be bound by both gene promoters and suppressors,
if one can discover a TF motif, then that gives some information about
the relationships between genes.
That is, two genes which share a TF motif will both be tend to be expressed
or inhibited in similar ways.
\end_layout
\begin_layout Standard
Specifically, I chose to explore the Saccharomyces cerevisiae genome
\begin_inset Foot
status collapsed
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as downloaded from http://hgdownload.cse.ucsc.edu/goldenPath/sacCer3/bigZips/
\end_layout
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[2] because 1) it is fully sequenced, 2) it is annotated with gene locations,
and 3) it is small enough for my limited computing resources to analyze.
S.
cerevisiae has about 6000 genes across 16 nuclear chromosomes, and slightly
over 12 million base pairs in total.
Of the 12 million base pairs, roughly 27% are outside known or suspected
genes coding regions, according to the UCSC genome annotations database.
\end_layout
\begin_layout Standard
There are three main portions to this report.
They are, in order, the method I used (section
\begin_inset CommandInset ref
LatexCommand ref
reference "sec:Method"
\end_inset
), results and discussion (section
\begin_inset CommandInset ref
LatexCommand ref
reference "sec:Results"
\end_inset
), and finally the tools and and techniques I developed to support my method
(section
\begin_inset CommandInset ref
LatexCommand ref
reference "sec:Tools-and-Techniques"
\end_inset
).
I close with thoughts about future work.
\end_layout
\begin_layout Section
Prior Work
\end_layout
\begin_layout Standard
The idea of computationally searching for motifs is not new.
One technique is to use expectation maximization (EM) to find sequences
that are highly likely to be motifs [3].
The EM method is not typically used for searches across an entire genome,
but for a selected set of sequences.
There are also databases of known motifs such as TRANSFAC (http://www.gene-
regulation.com/pub/databases.html, limited free functionality) and JASPAR
(http://jaspar.genereg.net/).
The Wikipedia page for 'Sequence Motif'
\begin_inset Foot
status collapsed
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http://en.wikipedia.org/wiki/Sequence_motif
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has an extensive list of software and databases related to motif finding.
\end_layout
\begin_layout Section
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name "sec:Method"
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Method
\end_layout
\begin_layout Standard
So we want to find which TF motifs are most prevalent on the S.
cerevisiae genome.
The naive approach is, for each length of motif desired, to convolve all
possible combinations of that length along the non gene coding regions
of some organism's genome.
A sequence of length N is considered a match at some position along the
genome if its convolution score there is sufficiently high.
Then, one simply records which test sequences result in the most 'hits'
along the genome.
\end_layout
\begin_layout Standard
Convolution here means the sum of matching nucleotides between some test
sequence and a position on the genome, where mismatches have a weight of
-1.
For example, the convolution of AAAA and AAAG has a score of
\begin_inset Formula $+3-1=2$
\end_inset
, and AAA convolved along AAGTC results in the sequence of scores
\begin_inset Formula $<1,-1,-3>$
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.
Unlike the traditional meaning convolution, I discard positions without
complete overlap.
Thus, for complete coverage of 10-mers in the case of S.
cerevisiae, approximately
\begin_inset Formula $4^{10}\cdot3.3\times10^{6}=$
\end_inset
3.3 trillion comparisons would have to be made.
This does not account for gaps, additions, elisions or other small variations
in the motif.
This is a combinatorial problem and so clearly
\emph on
the naive method is not scalable
\emph default
above a small number of base pairs.
\end_layout
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A key observation is that motifs can be represented by varying base sequences
that fulfill the same biological function.
That is, small changes in chemistry (e.g.
base pair substitution) may not radically effect the purpose of a given
motif.
Hence the naive approach also fails in that it would consider
\noun on
TATATT
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CAAATG, TATATTGAAATG and TATATTCAAA* to be different motifs even though
they very likely represent the same one, though the first two differ in
the 7th letter and the last is shorter by one base pair.
My solution to this dual problem of the naive approach is to use a modified
Needleman-Wunsch (mNW) algorithm, where no backtracking is done, instead
of plain convolution.
Further, I only use a randomly selected subset of all possible N-mers.
I accept a sequence as
\begin_inset Quotes eld
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good
\begin_inset Quotes erd
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via simple thresholding on the final score: a score of 9 or 10 was accepted
for the 10-mers under test, which allows for some wiggle-room in terms
of closeness and nucleotide variance among the same motif.
\end_layout
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This is a probabilistic approximation approach.
It is (hopefully) biologically sound because a large enough subset of test
sequences with an appropriate threshold covers a significant portion of
possible matches.
Even though the mNW algorithm is quadratic instead of linear in the test
sequence size, I do not have to test all sequences and also get positive
results for sequences that match against motifs of length 9 and 11 as well
as 10.
\end_layout
\begin_layout Standard
One of the main benefits of this approach, unlike the EM algorithm for motif
finding, is that it is trivially parallelized.
Testing each random guess is independent of all other guesses.
Thus, each mNW sweep through the non gene regions can be done separately
in its own process across networked machines and all results are accumulated
by another process.
For this project I had access to about 28 logical cores (14 without hyper-threa
ding).
Search speed is linear in the number of processing cores available.
\end_layout
\begin_layout Section
\begin_inset CommandInset label
LatexCommand label
name "sec:Results"
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Results and Discussion
\end_layout
\begin_layout Standard
My results come from 20,000 randomly generated test sequences of length
10.
At face value, 20,000 sequences only covers 2% of all length 10 sequences,
but as mentioned above my algorithm accounts for motifs that vary slightly
in their composition and so I believe this is sufficient to capture high
scoring motifs.
See Table
\begin_inset CommandInset ref
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reference "tab:Top-30-results"
\end_inset
and Figure
\begin_inset CommandInset ref
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reference "fig:Graph-of-top"
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for the top 30 scoring sequences that were found.
The top 10 sequences are visualized in Figure
\begin_inset CommandInset ref
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reference "fig:Visualization-of-top"
\end_inset
with a zoomed in section shown in Figure
\begin_inset CommandInset ref
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for example detail.
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<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
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\size footnotesize
TTTGCTTTTC
\end_layout
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</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
189
\end_layout
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</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
TTCAATTATT
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
AATAATTGAA
\end_layout
\end_inset
</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
174
\end_layout
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</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
TGTAAGAAAA
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
TTTTCTTACA
\end_layout
\end_inset
</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
170
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
AAACGTAAAA
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
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\size footnotesize
TTTTACGTTT
\end_layout
\end_inset
</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
167
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
TGTAAAATTA
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
TAATTTTACA
\end_layout
\end_inset
</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
165
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
CTTATTTTTG
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
CAAAAATAAG
\end_layout
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</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
163
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
ATTATCCTTT
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
AAAGGATAAT
\end_layout
\end_inset
</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
154
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
AAAAACATCA
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
TGATGTTTTT
\end_layout
\end_inset
</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
153
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
GGAGAAAAAT
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
ATTTTTCTCC
\end_layout
\end_inset
</cell>
</row>
<row>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text
\begin_layout Plain Layout
\family typewriter
\size footnotesize
143
\end_layout
\end_inset
</cell>
<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
\begin_inset Text