UGP Variant Pipeline 1.4.0

Utah Genome Project

Oct. 2015
Variant Calling Pipeline Version 1.4.0

Software Versions

• UGPp is a lightweight NGS pipeline, created for the
 Utah Genome Project (UGP)
• BWA: 0.7.10-r789
• GATK: 3.3-0-g37228af
• SamTools: 1.2-192-gfcaafe0 (using htslib 1.2.1-194-gf859e8d)
• Samblaster: Version 0.1.21
• Sambamba: v0.5.1
• FastQC: v0.11.2
• Tabix: 0.2.5 (r1005)
• WHAM: v1.7.0-160-g2a51

Data Source

Data sets used for the variant calling pipeline come from the Broad GSA (GATK) group as the 'GATK resource bundle version 2.8

wget -r ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/2.5/b37

Reference Genome (GRCh37) Now includes phix sequence.

• human_g1k_v37_decoy.fasta

Call region file generated from NCBI

• GRCh37 GFF3

VCF files for RealignerTargetCreator knowns and dbsnp for BaseRecalibrator.

• known_indel: Mills_and_1000G_gold_standard.indels.b37.vcf
• known_indel: 1000G_phase1.indels.b37.vcf
• known_dbsnp: dbsnp_137.b37.vcf

Background Files

• We have created 1000Genomes (BWA mem/GATK 3.0+) background files to be ran concurrently with the GenotypeGVCFs step.

Groups Currently completed:

• CEU
• GBR
• FIN

Version 1.0.5 background files have been updated to show only the indviduals of each group, not the file names.

This is a complete list of the background individuals for run completed > 1.0.5 [1]

If you would like a copy of the current files, we have made a public AWS s3 bucket
Using s3cmd execute the following command: 
s3cmd get s3://ugp-1k-backgrounds --recursive

Alternatively to access the files without s3cmd the following use the following URLs:
http://s3-us-west-2.amazonaws.com/ugp-1k-backgrounds/CEU_mergeGvcf.vcf
http://s3-us-west-2.amazonaws.com/ugp-1k-backgrounds/CEU_mergeGvcf.vcf.idx
http://s3-us-west-2.amazonaws.com/ugp-1k-backgrounds/FIN_mergeGvcf.vcf
http://s3-us-west-2.amazonaws.com/ugp-1k-backgrounds/FIN_mergeGvcf.vcf.idx
http://s3-us-west-2.amazonaws.com/ugp-1k-backgrounds/GBR_mergeGvcf.vcf
http://s3-us-west-2.amazonaws.com/ugp-1k-backgrounds/GBR_mergeGvcf.vcf.idx

Resource files for VariantRecalibrator_SNP

• hapmap_3.3.b37.vcf
• 1000G_omni2.5.b37.vcf
• 1000G_phase1.snps.high_confidence.b37.vcf

Resource files for VariantRecalibrator_INDEL

• Mills_and_1000G_gold_standard.indels.b37.vcf
• 1000G_phase1.indels.b37.vcf

Indexing

The following indexing is required using BWA, Picard and SamTools. GATK requires all three. However this step only needs to be done once "per-machine".

• BWA

bwa index -a bwtsw human_g1k_v37_decoy.fasta

• Picard

java -jar CreateSequenceDictionary.jar R=human_g1k_v37_decoy.fasta O=human_g1k_v37_decoy.dic

• SamTools

samtools faidx human_g1k_v37_decoy.fasta

Fasta Sequences

The fasta file currently used is (including decoys):

• human_g1k_v37_decoy.fasta

A list of headers can be found here [2]

FastQ File Analyses

fastqc --threads # -o <output dir> -f fastq fastq-file.fq.gz 2> fastqc_run.log-#

Alignment

Align reads to the genome with bwa.

The 'BWA-mem' program will find the reference coordinates of the input reads (independent of their mate-pair). The following parameters are those used by the 1KG project and GATK for aligning Illumina data.

bwa mem -R "read group" human_g1k_v37_decoy.fasta Sample1_L1_R1.fq Sample1_L1_R2.fq | samblaster --addMateTags |sambamba view -f bam -l 0 -S /dev/stdin | 
sambamba sort -m 50G -o *_#_sorted_Dedup.bam

"The --addMateTags has been added to samblaster for lumpy [3] compatibility."

BAM File Analyses

• samtools stats has now replaced idxstats.

samtools stats [sorted dedup bam files] > dedup-bamfile.stats

• flagstat

/scratch/ucgd/lustre/u0413537/software/samtools-0.1.19/

samtools flagstat _#_sorted_Dedup.bam > _#_sorted_Dedup.flagstat 2> flagstat.log-#

BAM Merging

• sambamba

Merging lane BAM files into single individual BAM file.

/scratch/ucgd/lustre/u0413537/software/sambamba/ 
sambamba merge  
--nthreads # 
individual_#_sorted_Dedup_merged.bam 
[individual_#_sorted_Dedup.bam files]