2020.10.12丨二代变异检测

• 基因或基因组上的差别
  • 基因转录翻译过程
  • 变异类型
• Calling SNPs(Index)
  • Overall procedure
    • fig
  • step.1 mapping
    • BWA(https://github.com/lh3/bwa)
    • 建立索引
      • bwa index hg19.fa
    • 比对
      • 
    • SAM Format(https://samtools.github.io/hts-specs/)
      • fig
      • 转换BAM格式
        • samtools view –b A.sam > A.bam
      • 排序
        • samtools sort –O BAM A.bam > A.sorted.bam
      • 建立索引（bam.bai）
        • samtools index A.sorted.bam
  • step.2 Remove duplicates
    • fig
    • GATK https://github.com/broadinstitute/gatk/releases
    • code 
      • gatk MarkDuplicates \-I A.sorted.bam \ # Input BAM from alignment-O A.dedup.bam \ #Output BAM-M A.marked_dup_metrics.txt # Output metrics
  • step.3 Call SNP for each sample
    • fig3
    • 3.1 Build index
      • Need two indicies:• .fai (from samtools index)• .dict (from gatk CreateSequenceDictionary)
      • samtools index hg19.fa #Creates hg19.fa.faigatk CreateSequenceDictionary \ #Creates reference.fa.dict–R hg19.fa
    • 3.2 Call SNPs for each sample using HaplotypeCaller
      • code
        • gatk HaplotypeCaller \-R hg19.fa \-I A.sorted.bam \-O A.raw.gvcf \-ERC GVCF \-ploidy 2 \ #modify based on species or sample pool
  • step.4 Combine GVCF from all the samples and genotype
    • gatk CombineGVCFs\-R hg19.fa \-O combine_variants.raw.gvcf \--variant A.raw.gvcf \--variant B.raw.gvcf \
    • gatk GenotypeGVCFs \-R hg19.fa \-O combine_variants.raw.vcf \--variant combine_variants.raw.gvcf \
    • GVCF vs VCF
      • GVCF A record for all sites (including non-variant sites)
      • VCF Only variant sites
    • 4.1 Obtaining SNP and filter
      • code
        • gatk SelectVariants \-R hg19.fa \-O combine_SNP.raw.vcf \--variant combine_variants.raw.vcf--select-type-to-include SNP
        • gatk VariantFiltration \-R hg19.fa \-O combine_SNP.filtered.vcf \--variant combine_SNP.raw.vcf \–-filter-name “snp_filter” \--filter-expression “QD < 2.0 || FS > 60.0 || SOR > 3.0 || MQ < 40.0 ||MQRankSum < -12.5”
    • 4.2 Obtaining Indel and filter
      • code
        • gatk SelectVariants \-R hg19.fa \-O combine_INDEL.raw.vcf \--variant combine_variants.raw.vcf--select-type-to-include INDEL 
        • gatk VariantFiltration \-R hg19.fa \-O combine_INDEL.filtered.vcf \--variant combine_INDEL.raw.vcf \–-filter-name “indel_filter” \--filter-expression “QD < 2.0 || FS > 200.0 || SOR > 10.0 || MQ < 40.0 ||MQRankSum < -12.5”
    • 4.3 (Rare SNP and call rate filter)
      • VCFtools
      • code
        • vcftools \--vcf combine_SNP.filtered.vcf \--max-missing 0.8 \--maf 0.05 \--minDP 4 \--out final.snp.vcf \
    • Visualize SNP and Indel on IGV
      • • Integrated Genomics Viewerhttp://software.broadinstitute.org/software/igv/
• Calling SV
  • SV types
    • fig
  • Manta https://github.com/Illumina/manta
  • code 
    • /path/to/configManta.py \--bam ../02.dedup/A.dedup.bam \--referenceFasta ../ref/hg19.fa \--runDir A_mantaA_manta/runWorkflow.py
  • fig
  • Manta VCF
•