官网文档：https://support.sentieon.com/manual/_downloads/Sentieon.pdf

DNAseq基本流程

Sentieon - 图1

1 realign- Realigner

nodup.bam -> realn.bam

  1.  sentieon driver \
  2.   -t NUMBER_THREADS \
  3.   -i NODUP_BAM \\
  4.   -r REFERENCE \
  5.   --algo Realigner \
  6.   -k MILLS_INDELS \
  7.   REALIGNED_BAM

2 recal(BQSR) - QualCal

realn.bam -> recal.table [+ recal.bam]

  1. # 仅生成recal.table [推荐]
  2. sentieon driver \
  3.   -t NUMBER_THREADS \
  4.   -i REALIGNED_BAM \
  5.   -r REFERENCE \
  6.   --algo QualCal \
  7.   -k MILLS_INDELS \
  8.   -k DBSNP \
  9.   RECAL_DATA.TABLE
  11. # 同时生成recal.bam
  12. sentieon driver \
  13.   -t NUMBER_THREADS \
  14.   -i REALIGNED_BAM \
  15.   -r REFERENCE \
  16.   --algo QualCal \
  17.   -k MILLS_INDELS \
  18.   -k DBSNP \
  19.   RECAL_DATA.TABLE \
  20.   --algo ReadWriter \
  21.   RECALIBRATED_BAM

3 Variant calling - Haplotyper / Genotyper

此步可分染色体进行，然后再用bcftools concat进行合并

方式1：使用Haplotyper算法，输入文件为 realn.bam + recal.table

  1. sentieon driver \
  2.   -t NUMBER_THREADS \
  3.   -r REFERENCE \
  4.   -i REALIGNED_BAM \
  5.   -q RECAL_DATA.TABLE \
  6.   --algo Haplotyper \
  7.   --emit_mode gvcf \
  8.   --emit_conf=10  \
  9.   --call_conf=10 \
  10.   [-d dbSNP] \
  11.   VARIANT_GVCF

方式2： 使用Genotyper算法，输入文件为recal.bam

  1. sentieon driver 
  2.   -t NUMBER_THREADS \
  3.   -r REFERENCE \
  4.   -i RECALIBRATED_BAM \
  5.   --algo Genotyper \
  6.   [-d dbSNP] \
  7.   --emit_mode gvcf \
  8.   VARIANT_GVCF

PS: 注意方式2中不能使用recal.bam+recal.table作为输入，否则会recal两次而导致错误结果

4 joint calling - GVCFtyper

s1.g.vcf.gz, s2.g.vcf.gz, … -> merged.vcf.gz

把一个或多个样本的GVCF合并成一个VCF文件, 输入文件可以通过-v指定

  1. sentieon driver \
  2.   -t NUMBER_THREADS \
  3.   -r REFERENCE \
  4.   --algo GVCFtyper \
  5.   -d DBSNP \
  6.   -v s1.g.vcf.gz \
  7.   -v s2.g.vcf.gz \
  8.   merge.vcf.gz

也可以直接在输出文件后面列上

  1. sentieon driver -r reference.fasta [driver_options] \
  2.   --algo GVCFtyper [algo_options] output.vcf[.gz] input.g.vcf[.gz] ...

5 VQSR - VarCal + ApplyVarCal

VQSR: Variant Quality Score Recalibration 使用一些可信的已知位点构建模型，对检出的变异位点进行过滤 merge.vcf.gz -> SNP VQSR -> Apply SNP VQSR -> INDEL VQSR -> Apply INDEL VQSR -> out.vcf.gz

  1.  # Step1: SNP VQSR
  2. sentieon driver \\
  3.     -t {vqsr_threads} \\
  4.     -r {reffasta} \\
  5.     --algo VarCal \\
  6.     --var_type SNP \\
  7.     --resource {1000g_phase1} \\
  8.     --resource_param 1000G,known=false,training=true,truth=false,prior=10.0 \\
  9.     --resource {1000g_omni} \\
  10.     --resource_param omni,known=false,training=true,truth=true,prior=12.0 \\
  11.     --resource {dbsnp} \\
  12.     --resource_param dbsnp,known=true,training=false,truth=false,prior=2.0 \\
  13.     --resource {hapmap} \\
  14.     --resource_param hapmap,known=false,training=true,truth=true,prior=15.0 \\
  15.     --annotation QD --annotation MQ --annotation MQRankSum \\
  16.     --annotation ReadPosRankSum --annotation FS \\
  17.     --tranches_file VCF/all.merged.snp.tranches \\
  18.     -v VCF/all.merged.vcf.gz \\
  19.     VCF/all.merged.snp.recal
  21. # Step2: Apply SNP VQSR
  22. sentieon driver \\
  23.     -t {vqsr_threads} \\
  24.     -r {reffasta} \\
  25.     --algo ApplyVarCal \\
  26.     --var_type SNP \\
  27.     --recal VCF/all.merged.snp.recal \\
  28.     --tranches_file VCF/all.merged.snp.tranches \\
  29.     -v VCF/all.merged.vcf.gz \\
  30.     VCF/all.merged.snp.recal.vcf.gz
  32. # Step3: INDEL VQSR
  33. sentieon driver \\
  34.     -t {vqsr_threads} \\
  35.     -r {reffasta} \\
  36.     --algo VarCal \\
  37.     --var_type INDEL \\
  38.     --resource {dbsnp} \\
  39.     --resource_param dbsnp,known=true,training=false,truth=false,prior=2.0 \\
  40.     --resource {mills_indels} \\
  41.     --resource_param Mills,known=false,training=true,truth=true,prior=12.0 \\
  42.     --annotation QD --annotation MQ \\
  43.     --annotation ReadPosRankSum --annotation FS \\
  44.     --tranches_file VCF/all.merged.snp.indel.tranches \\
  45.     -v VCF/all.merged.snp.recal.vcf.gz \\
  46.     VCF/all.merged.snp.indel.recal
  48. # Step4: Apply INDEL VQSR
  49. sentieon driver \\
  50.     -t {vqsr_threads} \\
  51.     -r {reffasta} \\
  52.     --algo ApplyVarCal \\
  53.     --var_type INDEL \\
  54.     --recal VCF/all.merged.snp.indel.recal \\
  55.     --tranches_file VCF/all.merged.snp.indel.tranches \\
  56.     -v VCF/all.merged.snp.recal.vcf.gz \\
  57.     VCF/all.merged.snp.indel.recal.vcf.gz