NextFlow_RD_Genomic

Description

A simple base Rare disease and germline genomics pipeline to test the effects of down-sampling on variant calling

Basic Overview

Using the NextFlow workflow software to run the following pipeline

Setup

To run the pipeline, we need to obtain

• A genome build (GRCh38) - provided by the Broad institute - REQUIRED

$ cd data/genome
$ wget https://storage.googleapis.com/gcp-public-data--broad-references/hg38/v0/Homo_sapiens_assembly38.fasta

• FastQ sample (for workflow development) - REQUIRED

$ cd ../samples
$ wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR151/003/SRR1518253/SRR1518253_1.fastq.gz && \
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR151/003/SRR1518253/SRR1518253_2.fastq.gz

• FASTQ for low coverage WGS - OPTIONAL

# Download the files using HTTPS
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR063/SRR063269/SRR063269_1.fastq.gz
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR063/SRR063279/SRR063279_2.fastq.gz
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR063/SRR063269/SRR063269_2.fastq.gz
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR063/SRR063277/SRR063277_2.fastq.gz
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR063/SRR063277/SRR063277_1.fastq.gz
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR063/SRR063279/SRR063279_1.fastq.gz

# Concatenate the R1 files into NA19750_R1
cat SRR063269_1.fastq.gz SRR063277_1.fastq.gz SRR063279_1.fastq.gz > NA19750_lc_wgs_R1.fastq.gz

# Concatenate the R2 files into NA19750_R2
cat SRR063269_2.fastq.gz SRR063277_2.fastq.gz SRR063279_2.fastq.gz > NA19750_lc_wgs_R2.fastq.gz

• FASTQ for NA19750 Exome - OPTIONAL

wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR071/SRR071178/SRR071178_1.fastq.gz
wget https://ftp.sra.ebi.ac.uk/vol1/fastq/SRR071/SRR071178/SRR071178_2.fastq.gz

For VQSR and BQSR download the following files and indexes into the relevant directory - REQUIRED

# 1000G Omni 2.5
wget -L -O 1000G_omni2.5.hg38.vcf.gz \
  "https://zenodo.org/records/18267841/files/1000G_omni2.5.hg38.vcf.gz?download=1"
wget -L -O 1000G_omni2.5.hg38.vcf.gz.tbi \
  "https://zenodo.org/records/18267841/files/1000G_omni2.5.hg38.vcf.gz.tbi?download=1"

# 1000G Phase 1 high-confidence SNPs
wget -L -O 1000G_phase1.snps.high_confidence.hg38.vcf.gz \
  "https://zenodo.org/records/18267841/files/1000G_phase1.snps.high_confidence.hg38.vcf.gz?download=1"
wget -L -O 1000G_phase1.snps.high_confidence.hg38.vcf.gz.tbi \
  "https://zenodo.org/records/18267841/files/1000G_phase1.snps.high_confidence.hg38.vcf.gz.tbi?download=1"

# HapMap 3.3
wget -L -O hapmap_3.3.hg38.vcf.gz \
  "https://zenodo.org/records/18267841/files/hapmap_3.3.hg38.vcf.gz?download=1"
wget -L -O hapmap_3.3.hg38.vcf.gz.tbi \
  "https://zenodo.org/records/18267841/files/hapmap_3.3.hg38.vcf.gz.tbi?download=1"

# dbSNP 138 (uncompressed VCF)
wget -L -O Homo_sapiens_assembly38.dbsnp138.vcf \
  "https://zenodo.org/records/18267841/files/Homo_sapiens_assembly38.dbsnp138.vcf?download=1"
wget -L -O Homo_sapiens_assembly38.dbsnp138.vcf.idx \
  "https://zenodo.org/records/18267841/files/Homo_sapiens_assembly38.dbsnp138.vcf.idx?download=1"

# Mills + 1000G gold standard indels
wget -L -O Mills_and_1000G_gold_standard.indels.hg38.vcf.gz \
  "https://zenodo.org/records/18267841/files/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz?download=1"
wget -L -O Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.tbi \
  "https://zenodo.org/records/18267841/files/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.tbi?download=1"

Samplesheet

For paired end alignment use the format

sampleID fastq_path Sex

sample1 /path/to/sample1_1.fastq.gz	/path/to/sample1_2.fastq.gz	1
sample2 /path/to/sample2_1.fastq.gz	/path/to/sample2_2.fastq.gz	2

and for single end or collapsed reads use

sampleID fastq_path Sex

sample1 /path/to/sample1.fastq.gz	1
sample2 /path/to/sample2.fastq.gz	2

Running the pipeline

# Using Docker
$ nextflow run -profile docker main.nf

# Using docker in singularity
$ nextflow run -profile singularity main.nf

Note: Refer to the nexrflow.config and nextflow_schema.json for parameter selection.

DNANexus applet setup (A local applet for basic testing)

• DNANexus Python Bindings Documentation
• Install the app

pip install -r requirements.txt

• Routine maintenance Periodically update dxpy

$ pip install --upgrade dxpy

DNANexus Tutorial

• Overview videos
• Developer tutorial

$ dx select <your-project-name>
$ dx build --nextflow

GRCh37 Download

wget https://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/p13.plusMT/hg19.p13.plusMT.fa.gz -O Homo_sapiens_assembly37.fasta.gz

Syphilis genome

wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/008/745/GCA_000008745.1_ASM874v1/GCA_000008745.1_ASM874v1_genomic.fna.gz -O syphilis.fa.gz

Concatenate gzipped files

zcat file1.gz file2.gz | gzip > concatenated_file.gz