https://gitpod.io/#https://github.com/renatopuga/somatico

Análise Somática

GATK 4 Mutect2 Somático

T12020 - Aula Prática

Pipeline

• GATK4 - Mutect2
• Gene JAK2
• Referência chr9
  • Sobre as versões do Genoma Humano: https://gatk.broadinstitute.org/hc/en-us/articles/360035890711-GRCh37-hg19-b37-humanG1Kv37-Human-Reference-Discrepancies#grch37
• Amostras:
  • WP043 (tumor)
  • WP044 (normal)
• https://gatk.broadinstitute.org/hc/en-us/articles/360035894731-Somatic-short-variant-discovery-SNVs-Indels-

Workflow

Converter BAM para FASTQ

samtools view -h -b /Volumes/Seagate\ Expansion\ Drive/data-lpfap10/projects/proadi/exome/bam/WP043/WP043.bam 9:5021937-5126899 | samtools bam2fq -1 tumor_R1.fq -2 tumor_R2.fq - 

samtools view -h -b /Volumes/Seagate\ Expansion\ Drive/data-lpfap10/projects/proadi/exome/bam/WP044/WP044.bam 9:5021937-5126899 | samtools bam2fq -1 normal_R1.fq -2 normal_R2.fq - 

Converter BAM para JAK2 BAM

• BAM para BAM

samtools view -h -b /Volumes/Seagate\ Expansion\ Drive/data-lpfap10/projects/proadi/exome/bam/WP043/WP043.bam 9:5021937-5126899 > tumor_JAK2.bam

samtools view -h -b /Volumes/Seagate\ Expansion\ Drive/data-lpfap10/projects/proadi/exome/bam/WP044/WP044.bam 9:5021937-5126899 > normal_JAK2.bam

• Gerar index do BAM (.BAI)

samtools index tumor_JAK2.bam 

samtools index normal_JAK2.bam 

af-only-gnomad.vcf.gz (apenas região JAK2)

• Header do VCF

zgrep -w "\#" af-only-gnomad.raw.sites.chr.vcf.gz > header

• Apenas Região do Gene JAK2

zgrep -w "^chr9" af-only-gnomad.raw.sites.chr.vcf.gz  | awk '$2>=5021937 && $2<=5126899' > JAK2.region

• Concatenar header + vcf

cat header JAK2.region > af-only-gnomad-chr9.vcf

• Compactar

bgzip af-only-gnomad-chr9.vcf

• Index do VCF

tabix -p vcf af-only-gnomad-chr9.vcf.gz 

Download da Referência - chr9

• Download

wget -c https://hgdownload.soe.ucsc.edu/goldenPath/hg19/chromosomes/chr9.fa.gz

• Descompactar

gunzip chr9.fa.gz 

BWA

Algoritmo de alinhamento.

BWA install

• BWA install (Mac)

brew install bwa

• BWA install (Ubuntu)

sudo apt-get install bwa

• BWA install (Docker)

docker pull comics/bwa

BWA index

• Index chr9

bwa index chr9.fa 

samtools

Samtools is a suite of programs for interacting with high-throughput sequencing data. It consists of three separate repositories:

samtools install

• samtools install (Mac)

brew install samtools 

• samtools install (Ubuntu)

sudo apt-get install samtools 

• samtools install (Docker)

docker pull biocontainers/samtools

samtools faidx e index

• samtools faidx

samtools faidx chr9.fa

• samtools index

samtools index tumor_JAK2.bam

samtools index normal_JAK2.bam

GATK4

Version: 4.2.2.0

Genome Analysis Toolkit - Variant Discovery in High-Throughput Sequencing Data. https://gatk.broadinstitute.org/

GATK4 install

GATK4 install Docker

docker pull broadinstitute/gatk:4.2.2.0

GATK4 install Mac e Linux

• Download

wget -c https://github.com/broadinstitute/gatk/releases/download/4.2.2.0/gatk-4.2.2.0.zip

• Descompactar

unzip gatk-4.2.2.0.zip 

• Testando gatk

./gatk-4.2.2.0/gatk

GATK4 .dict

./gatk-4.2.2.0/gatk CreateSequenceDictionary -R chr9.fa -O chr9.dict

GATK4 intervals

./gatk-4.2.2.0/gatk ScatterIntervalsByNs -R chr9.fa -O chr9.interval_list -OT ACGT

Mutect2

Call somatic SNVs and indels via local assembly of haplotypes

Mutect2 Tumor e Normal

./gatk-4.2.2.0/gatk Mutect2 -R chr9.fa -I tumor_JAK2.bam -I normal_JAK2.bam -normal WP044 --germline-resource af-only-gnomad-chr9.vcf.gz -O somatic.vcf.gz -L chr9.interval_list

Calcular Contaminação

GetPileupSummaries

Tabulates pileup metrics for inferring contamination

• GetPileupSummaries Tumor

./gatk-4.2.2.0/gatk GetPileupSummaries -I tumor_JAK2.bam -V af-only-gnomad-chr9.vcf.gz -L chr9.interval_list  -O tumor_JAK2.table

• GetPileupSummaries Normal

./gatk-4.2.2.0/gatk GetPileupSummaries -I normal_JAK2.bam -V af-only-gnomad-chr9.vcf.gz -L chr9.interval_list  -O normal_JAK2.table

CalculateContamination

Calculate the fraction of reads coming from cross-sample contamination

./gatk-4.2.2.0/gatk CalculateContamination -I tumor_JAK2.table -matched normal_JAK2.table -O contamination.table

FilterMutectCalls

Filter somatic SNVs and indels called by Mutect2

./gatk-4.2.2.0/gatk FilterMutectCalls -R chr9.fa -V somatic.vcf.gz --contamination-table contamination.table -O filtered.vcf.gz

Funcotator

Functional Annotator

• 30G Source Somatic (1s)

  • ftp://[email protected]/bundle/funcotator/funcotator_dataSources.v1.7.20200521s.tar.gz

• Download Funcotator

wget -c ftp://[email protected]/bundle/funcotator/funcotator_dataSources.v1.7.20200521s.tar.gz

• Descompactar

tar -zxvf funcotator_dataSources.v1.7.20200521s.tar.gz

• Anotar

./gatk-4.2.2.0/gatk Funcotator --data-sources-path funcotator_dataSources.v1.7.20200521s -O funcotator.maf --output-file-format MAF --ref-version hg19 --reference chr9.fa --variant filtered.vcf.gz