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SNPCalling.md

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Software version

  • bwa 0.7.17-r1188
  • biobambam 2.0.87
  • samtools 1.9
  • varscan v2.4.4
  • bcftools 1.9
  • tabix 1.9

SNP calling pipeline with NGS reads

  • align illumina reads to reference genome
bwa mem -M -R '@RG\\tID:sample_id\\tSM:mysample\\tLB:sample_lib\\tPL:ILM' \ 
  -t 24 ref.fa reads.R1.fq.gz reads.R2.fq.gz | \
  samtools view -Shb -@ 24 | \
  samtools sort -@ 24 -m 2G  -o mysample.bam -O BAM
  • marked duplicates with biobambam2
bammarkduplicates2 I=mysample.bam O=mysample.markedd_dup.bam M=mysample.markedd_up.metrics \
  markthreads=2  tmpfile=/tmp
  • samtools mpileup
samtools mpileup -B -f  ref.fa -o mysample.markedd_dup.mpileup mysample.markedd_dup.bam
  • call snp with varscan
varscan mpileup2snp mysample.markedd_dup.mpileup --p-value 0.05 --mysample-vcf 1 | \
  bcftools view -Oz -o mysample.markedd_dup.varscan.vcf.gz
  • build index for variants
tabix mysample.markedd_dup.varscan.vcf.gz