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LRII.6 Bioinformatics

LRII.6 Bioinformatics

Owned by Gregg Randolph
Last updated: Mar 21, 2022
2 min read

 

Assign reads and otus to samples:

Assign Reads:

/project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/rawdata salloc --account=microbiome -t 0-06:00 mkdir -p /gscratch/grandol1/LRII_LocAd2_3_16_22/rawdata cd /gscratch/grandol1/LRII_LocAd2_3_16_22/rawdata unpigz --to-stdout /project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/rawdata/LRII-5-RMJan22_S1_L001_R1_001.fastq.gz | split -l 1000000 -d --suffix-length=3 --additional-suffix=.fastq - LRII_LocAd2_3_16_22_R1_ ;unpigz --to-stdout /project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/rawdata/LRII-5-RMJan22_S1_L001_R2_001.fastq.gz | split -l 1000000 -d --suffix-length=3 --additional-suffix=.fastq - LRII_LocAd2_3_16_22_R2_ //project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/rawdata/run_parse_count_onSplitInput.pl cd /project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/rawdata ./run_splitFastq_fwd.sh ./run_splitFastq_rev.sh cd /project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/rawdata ./run_aggregate.sh

Process through to otus:

salloc --account=microbiome -t 0-06:00 cd /project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/tfmergedreads ./run_slurm_mergereads.pl cd /project/microbiome/data_queue/seq/LRII_LocAd2_3_16_22/otu ./run_slurm_mkotu.pl

Exploration of Data created so far:

From my understanding, Line 9 from above simply splits the raw data into equal sized files, but the total number of reads should remain constant.

cd /gscratch/grandol1/LRII_LocAd2_3_16_22/rawdata

wc -l LRII*

Should return 8x the number of paired end reads (2x for R1 and 4x for the structure of fastq files).

This returns: 33809712 total

Divided by 8: 4226214

 

Line 11 then reads through all of the split files and assigns each read to a sample (parsed), to PhiX or non target (phixOther), or a mid error (truemiderrors). The reads assigned to these should add up to the numbers above.

wc -l parsed*

Returns: 25644064 total

Divided by 8: 3205508 assigned to samples.

Assigned/Total (*100) = percent assigned: ~76%

The target for samples was 80%.

 

Things get more confusing with the phixOther and truemiderror files, because they do not appear to be true fastq files nor do they appear to be Fasta. So, I do not know how to count reads.

Blasting random lines from phixOther returns a mix of phiX and ‘uncultured bacterium 16S’. I see no way of disentangling this.

So, let us explore the results of lines 13 to 17. These should be found in /project/microbiome/data_queue/seq/loc_ad2/rawdata/sample_fastq/16S/locad2

and

/project/microbiome/data_queue/seq/loc_ad2/rawdata/sample_fastq/16S/LRII

For locad2:

wc -l locad2*

Returns: 18304944 total

The file formats appear the same as the “parsed*” files above.

Divided by 8: 2288118

For LRII:

wc -l LRII*

Returns: 7339120 total

Divided by 8: 917390

LRII + locad2:

2288118+917390= 3205508

Same as the parsed read count above.