bascalling simulated reads

[capoony]

Dear all,

fantastic software, many thanks!

I have a very naive question. After simulating reads for AmpliconSeq data, I would like to basecall the reads with guppy and then use these FASTQ reads to benchmark my downstream pipeline. Unfortunately, I do not get a single read back that passed the quality filtering What are the correct configs for guppy? I fear I am useing the wrong settings here:

######## load dependencies #######

module load ONT/guppy_6.2.1_gpu

######## run analyses #######

guppy_basecaller \
--input_path data_${name}/fast5_pass \
--config dna_r10.4.1_e8.2_400bps_sup.cfg \
--compress_fastq \
--save_path SUP \
-x "cuda:0"

Please find below also the first few lines of the summary file.

filename	read_id	run_id	batch_id	channel	mux	start_time	duration	num_events	passes_filtering	template_start	num_events_template	template_duration	sequence_length_template	mean_qscore_template	strand_score_template	median_template	mad_template	scaling_median_template	scaling_mad_template
Flow1_pass_c97995_1.fast5	002206d6-3aae-48ed-ad5a-2bf5528ba046	c979953ae6ed4445ad3de5a23d1f2a4c	0	2845	1	8.000000	1.643500	1314	FALSE	8.000000	1314	1.643500	659	5.047206	2.888203	101.321495	21.638645	101.321495	21.638645
Flow1_pass_c97995_1.fast5	004c33d0-d1d2-48f9-b0f8-0358f7d187cc	c979953ae6ed4445ad3de5a23d1f2a4c	0	1940	1	4.000000	1.365250	1092	FALSE	4.000000	1092	1.365250	484	7.027078	2.824660	100.736664	21.931059	100.736664	21.931059
Flow1_pass_c97995_1.fast5	007fb85d-538c-4f4d-8f2a-37c3e44fcfb8	c979953ae6ed4445ad3de5a23d1f2a4c	0	1855	1	8.000000	1.842250	1473	FALSE	8.000000	1473	1.842250	781	4.801814	2.935820	100.151840	22.369680	100.151840	22.369680

Thanks a lot,

Martin

[mattloose]

Hi,

I'm fairly certain that @Adoni5 will chim ein with some comments here, but icarust is not going to give you high quality base called data - the simulations are not precise - they are merely good enough to get back mappable data. As a consequence the quality filter is best ignored. I certainly wouldn't use the sup model unless you really want too.... it's not going to improve your data as the signals are not simulated in a way that we would expect them to perform well.

Icarust is designed to test adaptive sampling workflows and real time analysis but you shoudl not trust the qualities of the base calls.

I hope that helps.

Matt

[capoony]

Dear Matt, that indeed helps a lot, many thanks!

best, Martin