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BABS-aDNASeq

A Nextflow pipeline script for processing aDNASeq samples.

The pipeline was written by The Bioinformatics & Biostatistics Group in collaboration with The Ancient Genomics Lab @ The Francis Crick Institute.

nextflow: http://www.nextflow.io nextflow-quickstart: http://www.nextflow.io/docs/latest/getstarted.html#get-started

The Analysis Pipeline Flow

alt unistrap

Quick Start

To run an BABS-aDNASeq analysis you will need to complete the following steps. These are explained in more detail further down.

  1. Obtain BABS-aDNASeq files from GitHub.
  2. Install/load nextflow-0.32.0 or higher.
  3. Configure reference genome file paths (genome.yml).
  4. Configure environment profile if running software via a module system.
  5. Create a sample design file.
  6. Run nextflow pipeline.

Get BABS-aDNASeq Files

To obtain BABS-aDNASeq files run the following git command.

git clone https://github.com/crickbabs/BABS-aDNASeq

BABS-aDNASeq.nf The Nextflow script. BABS-aDNASeq Wrapper script to run an analysis. nextflow.config Main BABS-aDNASeq config file. conf/babs_profile.config Profile configuration for running the script @ The Crick. conf/genomes.config Genomes configuration file for defining reference data. conf/multiqc_config.yml Multiqc configuration used to generate integrated QC report.

Load Nextflow Module

If you are working within a module environment such as that at The Crick, load the nextflow module.

module purge module load nextflow/0.30.2

Sample Design File

Fastq files are specified in a csv design file with the following columns.

  column 1 : Individual ID
  column 2 : Sequencing library ID
  column 3 : full path to fastq file R1
  column 4 : full path to fastq file R2

Running an aDNASeq-ByBABS Analysis

BABS-aDNASeq --outdir ./ --design design.csv --profile babs --genome hg19 --resume

Output Directories & Files

Flow Details

Merge fastqs with the same library ID

Adapter trimming with SeqPrep

Adapter trimming and paired-end overlap consensus building. Only the overlap is saved here. Non-overlapping read-pairs are discarded.

https://github.com/jstjohn/SeqPrep

BWA

Consensus overlaps are aligned to the specified reference using BWA. BAM files with read groups are created.

https://github.com/lh3/bwa/

Duplicate Removal

Duplicate alignments are removed using Picard.

Variant Calling

VCFs are created using samtools mpileup. QC metrics are produced using bcftools stats.

Consensus Fasta

Ambiguity encoded consensus fasta files are produced using vcftools consensus.

Random Fasta

Random allele fasta files are produced using htsbox pileup -R.

https://github.com/lh3/htsbox

Merge BAM files

BAM files from the same individual are merged using samtools merge. Varient calling and QC ae carried out at both the library and individual level.

QC

Alignment QC is assessed using pmdtools and CollectWgsMetrics, CollectWgsMetricsWithNonZeroCoverage, CollectOxoGMetrics & CollectAlignmentSummaryMetrics from Picard. A QC report is generated using multiqc.

 https://github.com/pontussk/PMDtools
 https://github.com/broadinstitute/picard
 https://github.com/ewels/MultiQC

Credits

The BABS-aDNASeq nextflow pipeline was written and developed by Philip East & Pontus Skoglund.

The Bioinformatics & Biostatistics Group (BABS) @ The Francis Crick Institute. Ancient Genomics @ The Francis Crick Institute.

Licence

This project is licensed under the MIT License - see the LICENSE.md file for details.