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  • call indel and SNP by varscan

    Hi all,

    I met problem when I work on a 7M bacterium genome. I mapped 140x of paired-end reads (151x2) from illumina to a Pacbio reference genome by BWA mem. Then used samtool to get a mpileup file. Finally using varscan to call indel and SNP. Below is commands I used:

    map:
    $bwa mem -M -t 6 Pacbio_ref reads_1.fq reads_2.fq | samtools view -buS - | samtools sort - illumina_paired_map_pacbio.sorted
    $samtools index illumina_paired_map_pacbio.sorted.bam
    [/CODE]

    get mpileup
    Code:
    $samtools mpileup -f Pacbio_ref.fastq illumina_paired_map_pacbio.sorted.bam > mpileup_output
    call SNP
    Code:
    java -Xmx6g -Djava.io.tmpdir=temp -jar VarScan.v2.3.9.jar  \
    mpileup2snp mpileup_output \
    --min-coverage 20  \
    --min-reads2 2 \
    --min-avg-.01 \
    --p-value 0.05  \
    --output-vcf 1 > 05snp.vcf
    After that, I got only 268 SNP, similar work for indel but only 78 were found.

    I don't think those numbers should be so low. Would you please help to check if there is anything wrong on any step? I think maybe the parameters in BWA were not set correctly. Please help!

    Thanks!

  • #2
    Don't know if it can solve your problem but you can try to add -B option to samtools mpileup, as discussed here for example:
    Details on the samtools mpileup command, base alignment quality (BAQ), multi-sample calling, and other features.

    Discussion of next-gen sequencing related bioinformatics: resources, algorithms, open source efforts, etc

    Comment

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