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.
Assign taxonomy
salloc --account=microbiome -t 0-02:00 --mem=500G module load swset/2018.05 gcc/7.3.0 module load vsearch/2.15.1 vsearch --sintax zotus.fa --db /project/microbiome/users/grandol1/ref_db/gg_16s_13.5.fa -tabbedout LRII.sintax -sintax_cutoff 0.8
Output:
Reading file /project/microbiome/users/grandol1/ref_db/gg_16s_13.5.fa 100%
1769520677 nt in 1262986 seqs, min 1111, max 2368, avg 1401
Counting k-mers 100%
Creating k-mer index 100%
Classifying sequences 100%
Classified 4038 of 4042 sequences (99.90%)
Convert into useful form:
awk -F "\t" '{OFS=","} NR==1 {print "OTU_ID","SEQS","SIZE","DOMAIN","KINGDOM","PHYLUM","CLASS","ORDER","FAMILY","GENUS","SPECIES"} {gsub(";", ","); gsub("centroid=", ""); gsub("seqs=", ""); gsub("size=", ""); match($4, /d:[^,]+/, d); match($4, /k:[^,]+/, k); match($4, /p:[^,]+/, p); match($4, /c:[^,]+/, c); match($4, /o:[^,]+/, o); match($4, /f:[^,]+/, f); match($4, /g:[^,]+/, g); match($4, /s:[^,]+/, s); print $1, d[0]=="" ? "NA" : d[0], k[0]=="" ? "NA" : k[0], p[0]=="" ? "NA" : p[0], c[0]=="" ? "NA" : c[0], o[0]=="" ? "NA" : o[0], f[0]=="" ? "NA" : f[0], g[0]=="" ? "NA" : g[0], s[0]=="" ? "NA" : s[0] }' LRII.sintax > LRIItaxonomy.csv