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genome-analysis

Genome analysis pipeline that follows GATK best practices from Broad Institute

Data Pre-processing

Mapping to reference genome

Human reference genome downloaded from: http://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/

Index the files (not sure if necessary, but was running into a [E::bwa_idx_load_from_disk] fail to locate the index files error when I didn't)

$ bwa index hg19/ucsc.hg19.fasta.gz

Read groups example:
-R "@RG\tID:HG5H.2\tSM:045F16\tLB:library1\tPL:illumina\tPU:HG5HWDSXX.2"
tID, tPU are obtained from fastq files.
tSM is name of the fastq file.

Use bwa-mem to map to reference

$ bwa mem -t 4 -R "@RG\tID:HG5H.2\tSM:043F13\tLB:library1\tPL:illumina\tPU:HG5HWDSXX.2" hg19/ucsc.hg19.fasta.gz /data/compbio/melkebir/ElKebir/043F13_CGGCGTGA-GCGCCTGT_L002_R1_001.fastq /data/compbio/melkebir/ElKebir/043F13_CGGCGTGA-GCGCCTGT_L002_R2_001.fastq > 043F13.bam

Sort and index

samtools sort bamfile.bam -o bamfile.sorted.bam

samtools index bamfile.bam

Mark duplicates using gatk command line tools

gatk --java-options -Xmx12G MarkDuplicates -I 045F16.sorted.bam -O 045F16.dedup.bam -M 045F16.dedup.metrics.txt gatk --java-options "-Djava.io.tmpdir=/scratch/tmp -Xmx12G" MarkDuplicates -I 043F13.sorted.bam -O 043F13.dedup.bam -M 043F13.dedup.metrics.txt

base recallibrator

Get the following .vcf files and their corresponding index files (.dict, .idx) from GATK resource bundle: dbsnp_138.hg19.vcf Mills_and_1000G_gold_standard.indels.hg19.sites.vcf 1000G_phase1.indels.hg19.sites.vcf

(dbsnp and 1000G were also avaialble in original UCSC ftp server, but I got them all from GATK bundle for consistency)

gunzip and IndexFeatureFiles for dbsnp_138.hg19.vcf (to solve block compression format error...see here: https://gatkforums.broadinstitute.org/gatk/discussion/11908/variantrecalibrator-error-using-gatk-bundle-vcf-files)

Deviation from Octopus paper: the Octopus paper uses .vcf.gz files but we used unzipped .vcf files because of block compression error, and GATK doesnt support zipped files (GATK documentation also does it this way)

Variant calling using octopus

Use script run_octopus.sh. --fast sacrifices some accuracy for speed.

Clarifications

  • Difference between bcftools and vcftools