Varscan2 Call snp_indel

最新推荐文章于 2024-10-31 19:49:25 发布

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VarScan2 的最新版本引入了一个新的子命令，用于按变异类型（体细胞、生殖细胞、LOH）和信心级别（高信心、低信心）隔离变异调用。这将生成四个输出文件，分别包含高信心和低信心的体细胞变异，生殖细胞变异以及LOH位点。

Varscan2官方文档

01:mpileup文件准备
samtools mpileup -d 1000 -Q 20 -q 30 -f /data2/references/Homo_sapiens/hg38.genomic.fa pa01_tumor1.bam >pa01_tumor1.mpileup

02:使用Varscan2的 somatic 命令
USAGE: java -jar VarScan.jar somatic [normal_pileup] [tumor_pileup] [output] 
OPTIONS
        normal_pileup - The SAMtools pileup file for Normal
        tumor_pileup - The SAMtools pileup file for Tumor
        output - Output base name for SNP and indel output

03:somatic 命令 可选参数
OPTIONS:
	--output-snp - Output file for SNP calls [default: output.snp]
	--output-indel - Output file for indel calls [default: output.indel]
	--min-coverage - Minimum coverage in normal and tumor to call variant [8]
	--min-coverage-normal - Minimum coverage in normal to call somatic [8]
	--min-coverage-tumor - Minimum coverage in tumor to call somatic [6]
	--min-var-freq - Minimum variant frequency to call a heterozygote [0.10]
	--min-freq-for-hom	Minimum frequency to call homozygote [0.75]
	--normal-purity - Estimated purity (non-tumor content) of normal sample [1.00]
	--tumor-purity - Estimated purity (tumor content) of tumor sample [1.00]
	--p-value - P-value threshold to call a heterozygote [0.99]
	--somatic-p-value - P-value threshold to call a somatic site [0.05]
	--strand-filter - If set to 1, removes variants with >90% strand bias
	--validation - If set to 1, outputs all compared positions even if non-variant

Variant Calling and Comparison

If tumor matches normal: 
        If tumor and normal match the reference 
            ==> Call Reference 
        Else tumor and normal do not match the reference 
            ==> Call Germline 

    Else tumor does not match normal: 
    Calculate significance of allele frequency difference by Fisher's Exact Test 
        If difference is significant (p-value < threshold):
            If normal matches reference
                ==> Call Somatic
            Else If normal is heterozygous
                ==> Call LOH
            Else normal and tumor are variant, but different
                ==> Call IndelFilter or Unknown
        Else difference is not significant:
        Combined tumor and normal read counts for each allele. Recalculate p-value.
            ==> Call Germline

variant_p_value:Variant p-value for Germline events somatic_p_value:Somatic p-value for Somatic/LOH events

Isolating Calls by Type and Confidence

The latest release of VarScan includes a new (undocumented) subcommand that will separate a somatic output file by somatic_status (Germline, Somatic, LOH). Somatic mutations will further be classified as high-confidence (.hc) or low-confidence (.lc).

The command: java -jar VarScan.jar processSomatic [output.snp]

The above command will produce 4 output files:
output.snp.Somatic.hc (high-confidence Somatic mutations)
output.snp.Somatic.lc (low-confidence Somatic mutations)
output.snp.Germline (sites called Germline)
output.snp.LOH (sites called loss-of-heterozygosity, or LOH)