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Aula_somaticoEP1

OBS.: Para todo esse papeline, utilizar o GitPod no terminal Bash

Referências:

(https://github.com/renatopuga/somaticoEP1/blob/main/README.md)

(https://github.com/renatopuga/somaticoEP2)

Instalando ferramentas

brew install sratoolkit

pip install parallel-fastq-dump

Download do arquivo a ser utilizado

wget -c https://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/3.0.0/sratoolkit.3.0.0-ubuntu64.tar.gz
tar -zxvf sratoolkit.3.0.0-ubuntu64.tar.gz
export PATH=$PATH://workspace/somaticoEP1/sratoolkit.3.0.0-ubuntu64/bin/
echo "Aexyo" | sratoolkit.3.0.0-ubuntu64/bin/vdb-config

time parallel-fastq-dump --sra-id SRR8856724 --threads 4 --outdir ./ --split-files --gzip

Fazendo o download das Referências do Genoma hg38 (FASTA, VCFs)

wget -c https://storage.googleapis.com/gatk-best-practices/somatic-hg38/af-only-gnomad.hg38.vcf.gz

wget -c https://storage.googleapis.com/gatk-best-practices/somatic-hg38/af-only-gnomad.hg38.vcf.gz.tbi

wget -c https://storage.googleapis.com/gatk-best-practices/somatic-hg38/1000g_pon.hg38.vcf.gz

wget -c https://storage.googleapis.com/gatk-best-practices/somatic-hg38/1000g_pon.hg38.vcf.gz.tbi

wget -c https://storage.googleapis.com/gatk-best-practices/somatic-hg38/small_exac_common_3.hg38.vcf.gz

wget -c https://storage.googleapis.com/gatk-best-practices/somatic-hg38/small_exac_common_3.hg38.vcf.gz.tbi

Download do exoma

wget -c https://hgdownload.soe.ucsc.edu/goldenPath/hg38/chromosomes/chr9.fa.gz

Instalando o BWA no gitpod

brew install bwa

Descompactando arquivo e indexando

gunzip chr9.fa.gz

bwa index chr9.fa

Instalando Samtools faidx e gerando arquivo

brew install samtools

samtools faidx chr9.fa

Convertendo FASTQ para BAM

NOME=WP312; Biblioteca=Nextera; Plataforma=illumina;

bwa mem -t 16 -M -R "@RG\tID:$NOME\tSM:$NOME\tLB:$Biblioteca\tPL:$Plataforma" \
chr9.fa \
SRR8856724_1.fastq.gz \
SRR8856724_2.fastq.gz > WP312.sam

Ao final desta etapa vc deverá ter esses aquivos e diretório:

ls

1 EP1

organizando os arquivos com samtools: fixmate, sort e index

time samtools fixmate -@10 WP312.sam WP312.bam
time samtools sort -O bam -@6 -m2G -o WP312_sorted.bam WP312.bam
time samtools index WP312_sorted.bam
time samtools rmdup WP312_sorted.bam WP312_sorted_rmdup_F4.bam
time samtools index WP312_sorted_rmdup_F4.bam

Instalando a ferramenta bedtools

brew install bedtools

Gerando o arquivo BED

bedtools bamtobed -i WP312_sorted_rmdup_F4.bam > WP312_sorted_rmdup.bed
bedtools merge -i WP312_sorted_rmdup.bed > WP312_sorted_rmdup_merged.bed
bedtools sort -i WP312_sorted_rmdup_merged.bed > WP312_sorted_rmdup_merged_sorted.bed

Ao final desta etapa vc deverá ter esses aquivos e diretório:

ls

2 EP1

Cobertura Média

git clone https://github.com/circulosmeos/gdown.pl.git
./gdown.pl/gdown.pl  https://drive.google.com/file/d/1pTMpZ2eIboPHpiLf22gFIQbXU2Ow26_E/view?usp=drive_link WP312_sorted_rmdup_F4.bam
./gdown.pl/gdown.pl  https://drive.google.com/file/d/10utrBVW-cyoFPt5g95z1gQYQYTfXM4S7/view?usp=drive_link WP312_sorted_rmdup_F4.bam.bai

bedtools coverage -a WP312_sorted_rmdup_merged_sorted.bed \
-b WP312_sorted_rmdup_F4.bam -mean \
> WP312_coverageBed.bed

Realizando filtro no arquivo gerado (total de reads >=20)

cat WP312_coverageBed.bed | \
awk -F "\t" '{if($4>=20){print}}' \
> WP312_coverageBed20x.bed

Instalando GATK4

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

Gerando o arquivo .dict

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

Gerando o arquivo interval_list

./gatk-4.2.2.0/gatk BedToIntervalList -I WP312_coverageBed20x.bed \
-O WP312_coverageBed20x.interval_list -SD chr9.dict

Convertendo Bed para Interval_list

-O WP312_coverageBed20x.interval_list -SD chr9.dict

Ao final desta etapa vc deverá ter esses aquivos e diretórios:

ls

3 EP1

Calculando contaminação

./gatk-4.2.2.0/gatk GetPileupSummaries \
	-I WP312_sorted_rmdup_F4.bam  \
	-V af-only-gnomad.hg38.vcf.gz \
	-L WP312_coverageBed20x.interval_list \
	-O WP312.table

./gatk-4.2.2.0/gatk CalculateContamination \
-I WP312.table \
-O WP312.contamination.table

Visualizando resultado

cat WP312.contamination.table

MuTect2 Call

./gatk-4.2.2.0/gatk Mutect2 \
  -R chr9.fa \
  -I WP312_sorted_rmdup_F4.bam \
  --germline-resource af-only-gnomad.hg38.vcf.gz  \
  --panel-of-normals 1000g_pon.hg38.vcf.gz \
  -L WP312_coverageBed20x.interval_list \
  -O WP312.somatic.pon.vcf.gz

MuTect2 FilterMutectCalls

./gatk-4.2.2.0/gatk FilterMutectCalls \
	-R chr9.fa \
	-V WP312.somatic.pon.vcf.gz \
	--contamination-table WP312.contamination.table \
	-O WP312.filtered.pon.vcf.gz

Ao final desta etapa vc deverá ter esses aquivos e diretórios:

ls

4 EP1

Visualizando resultado

less -SN WP312.filtered.pon.vcf.gz

Retornando

q

Quantas variantes têm no arquivo gerado?

zgrep -c ^chr9 WP312.filtered.pon.vcf.gz

Quantas variantes passaram no filtro?

zgrep ^chr9 WP312.filtered.pon.vcf.gz | grep -c PASS

Começando uma nova etapa

mkdir hg38
mv * hg38

Aula somatico EP2

Download dos arquivos de referência hg19

wget -c https://storage.googleapis.com/gatk-best-practices/somatic-b37/Mutect2-WGS-panel-b37.vcf
wget -c https://storage.googleapis.com/gatk-best-practices/somatic-b37/Mutect2-WGS-panel-b37.vcf.idx
wget -c  https://storage.googleapis.com/gatk-best-practices/somatic-b37/af-only-gnomad.raw.sites.vcf
wget -c  https://storage.googleapis.com/gatk-best-practices/somatic-b37/af-only-gnomad.raw.sites.vcf.idx

Cobertura

mover arquivos necessários

cd hg38
mv WP312_sorted_rmdup_F4.bam /workspace/somaticoEP2
mv WP312_sorted_rmdup_merged_sorted.bed /workspace/somaticoEP2

brew install bedtools
bedtools bamtobed -i WP312_sorted_rmdup_F4.bam > WP312_sorted_rmdup.bed
bedtools merge -i WP312_sorted_rmdup.bed > WP312_sorted_rmdup_merged.bed
bedtools sort -i WP312_sorted_rmdup_merged.bed > WP312_sorted_rmdup_merged_sorted.bed

bedtools coverage -a WP312_sorted_rmdup_merged_sorted.bed \
-b WP312_sorted_rmdup_F4.bam -mean \
> WP312_coverageBed.bed

Para gerar o bed de cobertura 20X

cat WP312_coverageBed.bed | \
awk -F "\t" '{if($4>=20){print}}' \
> WP312_coverageBed20x.bed

Adicionando o chr nos arquivos de referência

grep "\#" af-only-gnomad.raw.sites.vcf > af-only-gnomad.raw.sites.chr.vcf
grep  "^9" af-only-gnomad.raw.sites.vcf |  awk '{print("chr"$0)}' >> af-only-gnomad.raw.sites.chr.vcf
bgzip af-only-gnomad.raw.sites.chr.vcf
tabix -p vcf af-only-gnomad.raw.sites.chr.vcf.gz

grep "\#" Mutect2-WGS-panel-b37.vcf > Mutect2-WGS-panel-b37.chr.vcf 
grep  "^9" Mutect2-WGS-panel-b37.vcf |  awk '{print("chr"$0)}' >> Mutect2-WGS-panel-b37.chr.vcf 
bgzip Mutect2-WGS-panel-b37.chr.vcf 
tabix -p vcf Mutect2-WGS-panel-b37.chr.vcf.gz

Movendo os arquivos GATK para o diretório em execução

cd hg38
mv gatk-4.2.2.0 /workspace/somaticoEP2
mv gatk-4.2.2.0.zip /workspace/somaticoEP2

Geraldo o arquivo .dict e interval

./gatk-4.2.2.0/gatk CreateSequenceDictionary -R chr9.fa -O chr9.dict
./gatk-4.2.2.0/gatk ScatterIntervalsByNs -R chr9.fa -O chr9.interval_list -OT ACGT
./gatk-4.2.2.0/gatk BedToIntervalList -I WP312_coverageBed20x.bed \
-O WP312_coverageBed20x.interval_list -SD chr9.dict

Calculando contaminação

gzip -k af-only-gnomad.raw.sites.chr.vcf
./gatk-4.2.2.0/gatk GetPileupSummaries \
	-I WP312_sorted_rmdup_F4.bam  \
	-V af-only-gnomad.raw.sites.chr.vcf.gz  \
	-L WP312_coverageBed20x.interval_list \
	-O WP312.table

 ./gatk-4.2.2.0/gatk CalculateContamination \
-I WP312.table \
-O WP312.contamination.table

Visualizando contaminação

cat  WP312.contamination.table

GATK Mutect2

OBS.: Esta parte demorará um pouco mais que o normal!ls

 ./gatk-4.2.2.0/gatk Mutect2 \
  -R chr9.fa \
  -I WP312_sorted_rmdup_F4.bam \
  --germline-resource af-only-gnomad.raw.sites.chr.vcf.gz  \
  --panel-of-normals Mutect2-WGS-panel-b37.chr.vcf.gz \
  --disable-sequence-dictionary-validation \
  -L WP312_coverageBed20x.interval_list \
  -O WP312.somatic.pon.vcf.gz

  ./gatk-4.2.2.0/gatk FilterMutectCalls \
-R chr9.fa \
-V WP312.somatic.pon.vcf.gz \
--contamination-table WP312.contamination.table \
-O WP312.filtered.pon.vcf.gz

Agora vamos comparar os VCFs gerados com os VCFs, WP312.filtered.vcf e WP312.filtered.vcf.gz do link abaixo:

(https://drive.google.com/drive/folders/1m2qmd0ca2Nwb7qcK58ER0zC8-1_9uAiE)

zgrep "\#" WP312.filtered.vcf.gz > header.txt
zgrep -v "\#" WP312.filtered.vcf.gz | awk '{print("chr"$0)}' > variants.txt
cat header.txt variants.txt > WP312.filtered.chr.vcf
bgzip WP312.filtered.chr.vcf
tabix WP312.filtered.chr.vcf.gz

zgrep "^\#\|chr9" WP312.filtered.chr.vcf.gz > WP312.filtered.chr9.vcf
bgzip  WP312.filtered.chr9.vcf
tabix WP312.filtered.chr9.vcf.gz

brew install vcftools

Realizando a comparação

vcf-compare WP312.filtered.pon.vcf.gz WP312.filtered.chr9.vcf.gz

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