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htslib官网上给的一个WGS/WES的流程。关于htslib、samtools和bcftools之间的关系，可以在sanger官网查看其解释：

HTSlib is a software library for manipulating various sequencing and variant file formats: SAM, BAM, CRAM, VCF, and BCF. SAMtools and BCFtools are applications built around HTSlib, performing format conversion, file merging and splitting, sorting, variant calling, and much more.

workflow主要三步骤：

• Mapping

• Improvement

• Variant Calling

  bwa index
  bwa mem -R '@RG\tID:foo\tSM:bar\tLB:library1' > lane.sam #官方给的，我认为是read1.fq和read2.fq
  samtools fixmate -O bam
  samtools sort -O bam -o -T

  realign gapped alignment

  java -Xmx2g -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R -I -o --known
  java -Xmx4g -jar GenomeAnalysisTK.jar -T IndelRealigner -R -I -targetIntervals --known -o

  BQSR

  ava -Xmx4g -jar GenomeAnalysisTK.jar -T BaseRecalibrator -R -knownSites >bundle/b38/dbsnp_142.b38.vcf> -I -o
  java -Xmx2g -jar GenomeAnalysisTK.jar -T PrintReads -R -I --BSQR -o

  #MarkDuplicates
  java -Xmx2g -jar MarkDuplicates.jar VALIDATION_STRINGENCY=LENIENT INPUT= INPUT= INPUT= OUTPUT=

  samtools merge
  samtools index

  realign your INDELS（可选）

  java -Xmx2g -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R -I -o --known >bundle/b38/Mills1000G.b38.vcf>
  java -Xmx4g -jar GenomeAnalysisTK.jar -T IndelRealigner -R -I -targetIntervals --known >bundle/b38/Mills1000G.b38.vcf> -o

  samtools index

  bcftools mpileup -Ou -f | bcftools call -vmO z -o

  exam bcf（可选）

  bcftools mpileup -Ob -o -f
  bcftools call -vmO z -o

  tabix -p vcf

  bcftools stats -F -s - >
  mkdir plots
  plot-vcfstats -p plots/

  bcftools filter -O z -o -s LOWQUAL -i'%QUAL>10'