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Raw reads

We received two files with sequence reads.

Demultiplexing

mkdir /gscratch/grandol1/1SaugOdds /gscratch/grandol1/1SaugOdds/rawreads

cd /gscratch/grandol1/1SaugOdds
unpigz --to-stdout /project/gtl/data/distribution/Wagner/SJohnson/1Saug/rawreads/1SaugOdds.fastq.gz | split -l 16000000 -d --suffix-length=3 --additional-suffix=.fastq - 1SaugOdds_
/project/gtl/data/distribution/Wagner/SJohnson/1Saug/demultiplex/run_parsebarcodes_onSplitInput_Odds.pl

Repeat for Evens

mkdir /gscratch/grandol1/1SaugEvens /gscratch/grandol1/1SaugEvens/rawreads

cd /gscratch/grandol1/1SaugEvens
unpigz --to-stdout /project/gtl/data/distribution/Wagner/SJohnson/1Saug/rawreads/1SaugEvens.fastq.gz | split -l 16000000 -d --suffix-length=3 --additional-suffix=.fastq - 1SaugEvens_
/project/gtl/data/distribution/Wagner/SJohnson/1Saug/demultiplex/run_parsebarcodes_onSplitInputEvens.pl

I modified the script from 16S/ITS work for splitting fastq files based on information in their info line, to different files. It is: /project/gtl/data/distribution/Wagner/SJohnson/1Saug/splitFastq_manyInputfiles_gbs.pl and is run with run_splitFastq_gbs.sh, in the same directory. Output is in /project/gtl/data/distribution/Wagner/SJohnson/1Saug/demultiplex.

  • compressed all sample_fastq/ files with pigz: using sbatch /project/microbiome/data/seq/HMAX1/demultiplex/run_pigz.sh

  • moved fastq for all four blank samples (data are all in one file because names are collapsed; noted above) to a subfolder (/project/microbiome/data/seq/HMAX1/demultiplex/sample_fastq/blanks), to get them out of the way.

  • started denovo assembly in /gscratch/buerkle/data/HMAX1/denovo Completed first step for dDocent and am running cd-hit for 92%, 96% and 98% minimum match. Initially didn’t give these enough wall time and in reruns I bumped up the number of cores to 16.

Assembly

Working in /project/microbiome/data/seq/HMAX1/assem and assembling all reads in /project/microbiome/data/seq/HMAX1/demultiplex/sample_fastq/ against /project/evolgen/data/public/genomes/helianthus/GCF_002127325.2_HanXRQr2.0-SUNRISE_genomic.fna.

  1. Ran bwa index -a bwtsw GCF_002127325.2_HanXRQr2.0-SUNRISE_genomic.fna by hand in an interactive node (took roughly one hour)

  2. Commands are in 0_assem.nf. Run this with nextflow run -bg 0_assem.nf -c teton.config. These are jobs are using: module load swset/2018.05 gcc/7.3.0 bwa/0.7.17 samtools/1.12 as specified in teton.config in this directory (bwa is version 0.7.17-r1188). Output is in /project/microbiome/data/seq/HMAX1/assem/sambam/. Gave each job 60 minutes, which was unnecessarily long, but conservative. Longest running jobs I could see were less than 20 minutes. Moved all 468 inputs files through in about 30 minutes total.

  3. I removed the duplicative sam and unsorted bam files with: rm -f *.sam *[^d].bam, saving ~270 GB of space

Variant calling

  • Following steps from https://github.com/zgompert/DimensionsExperiment.

  • Built bcftools version 1.16 and installed in /project/evolgen/bin/.

  • bcftools needed reference genome in bzip2 format, not gzip. So I now simply have an unzipped reference genome, which I have reindexed.

  • Completed this step with something like: sbatch --account=evolgen --time=1-00:00 --nodes=1 --mem=8G --mail-type=END  0_call_variants.sh (this took 12 hours and 40 minutes and 552 MB of RAM; I asked for 120GB, which likely gave me the whole node and made it a bit faster)

  • Filtered vcf with 1_filter_variants.sh, which contains notes on the criteria that I used (could be altered to suit). This set is based on a fairly tight set of criteria (it matches what we used for a recent paper), which could be modified as needed. Note that currently there is no explicit minor allele frequency filtering. There are 5016 sites in hmax_variants_filtered.vcf

    • ## minimum mapping quality of 30 was already enforced in bcftools mpileup

## These are written as exclusion filters
# ------ INFO/DP < 952
# 2x depth overall: obtained with INFO/DP > 951 (476 x 2 = 952)
##INFO=<ID=DP,Number=1,Type=Integer,Description="Raw read depth">

# ------ INFO/AC1 < 10 
## a minimum of 10 alt reads to support a polymorphism

# ------ INFO/BQBZ > 1e-5
##INFO=<ID=BQBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Base Quality Bias (closer to 0 is better)">

# ------ INFO/RPBZ > 1e-5
##INFO=<ID=RPBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Read Position Bias (closer to 0 is better)">

## biallelic snps obtained in bcftools view

## 476 individuals (80% with data would mean 380 individuals; 380/476=0.798)
## set bcftools view to include only sites with the fraction of missingness less than 0.2

To do:

  • Summarize the parse report files in /gscratch with some code to iterate over all the individual reports and get an overall count.

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