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Old 11-27-2015, 03:13 AM   #1
doublealice
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Location: US

Join Date: Feb 2011
Posts: 24
Default 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!
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Old 12-03-2015, 08:32 AM   #2
Jane M
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Location: Paris

Join Date: Aug 2011
Posts: 237
Default

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:
http://massgenomics.org/2012/03/5-th...s-mpileup.html
http://seqanswers.com/forums/showthr...light=varscan2
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