1Sauger bioinformatics

Raw reads

We received two files with sequence reads.

Demultiplexing

Repeat for Evens

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/gtl/data/distribution/Wagner/SJohnson/1Saug/demultiplex/run_pigz.sh

• started denovo assembly in /gscratch/grandol1/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.

Stopped here 10-23-2024:

Assembly

Sep 10, 2022 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