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TronFlow Copy Number Calling

GitHub tag (latest SemVer) DOI Run tests License Powered by Nextflow run with conda

The TronFlow Copy Number Calling pipeline is part of a collection of computational workflows for tumor-normal pair somatic variant calling. Discover other TronFlow workflows at the TRON-Bioinformatics GitHub page.

Find the documentation here Documentation Status

Introduction

TronFlow Copy Number Calling is a workflow created to detect copy number variations (CNVs) on whole-exome sequencing data from human tumor-normal pairs.

The pipeline is built using Nextflow, a workflow management tool that "enables scalable and reproducible scientific workflows using software containers" (Di Tommaso et al., 2017). In particular, Nextflow's DSL2 syntax extension is applied which enables the usage of module libraries and therefore eases the process of creating complex data analysis pipelines. Taking advantage of this feature, the pipeline re-uses modular Nextflow implementations from nf-core/modules (Ewels et al., 2020) if available. Furthermore, the pipeline employs a comprehensive collection of test datasets from nf-core/test-datasets for continuous integration tests.

Pipeline summary

This workflow integrates the following tools for somatic copy number calling based on tumor-normal pairs:

  • CNVkit (Talevich et al., 2016)
  • Sequenza (Favero et al., 2015)
  • More coming soon...

A selection of one (or multiple) tool(s) can be achieved using the --tools flag. For more information about the pipeline's usage, please continue reading here.

Usage

Software requirements

Minimum requirements:

  • java >= 11: We recommend using SDKMAN! for installation of the Java SDK. See also here for nextflow specific instructions.
  • nextflow >= 19.10.0: We recommend using the latest stable release of nextflow. For installation instructions please see also here.
  • conda: We recommend using mamba as a faster alternative.

Input table

First, create a samplesheet with your input data that complies to the following format:

input_files.tsv:

sample_1<TAB>/path/to/sample_1_tumor.bam<TAB>/path/to/sample_1_normal.bam
sample_2<TAB>/path/to/sample_2_tumor_1.bam,/path/to/sample_2_tumor_2.bam<TAB>/path/to/sample_2_normal_1.bam,/path/to/sample_2_normal_2.bam

Note The pipeline expects a file containing three tab-separated columns without a header. Each row represents a pair of tumor and normal BAM files. Multiple tumor or normal BAM files (i.e. replicates) can be provided separated by commas. The BAM files need to be sorted and indexed.

How to run the pipeline

Option 1: Run it from GitHub as follows...

$ nextflow run tron-bioinformatics/tronflow-copy-number-calling -r <RELEASE|BRANCH> -profile conda --input_files <YOUR_INPUT_FILE> --reference <YOUR_REFERENCE_FASTA> --intervals <YOUR_TARGET_REGIONS_BED> --tools cnvkit,sequenza

Option 2: Download the project and run it as follows...

$ nextflow run main.nf -profile conda --input_files <YOUR_INPUT_FILE> --reference <YOUR_REFERENCE_FASTA> --intervals <YOUR_TARGET_REGIONS_BED> --tools cnvkit,sequenza

See help message for all of the available options when running the pipeline:

$ nextflow run main.nf --help

TronFlow Copy Number Calling <VERSION>

Author: <AUTHORS>
DOI: <DOI>
GitHub: https://github.com/TRON-Bioinformatics/tronflow-copy-number-calling

Nextflow pipeline for copy number calling using different tools

Usage:
    nextflow run main.nf -profile conda --input_files input_files.tsv --reference reference.fasta --intervals target_region.bed --tools cnvkit,sequenza

Input:
    * input_files: the path to a tab-separated values file containing in each row the sample name, tumor bam and normal bam
    The input file does not have header!
    Example input file:
    name1	tumor_bam1	normal_bam1
    name2	tumor_bam2	normal_bam2
    * reference: path to the FASTA genome reference
    * intervals: path to the BED file with the targeted region
    * tools: tools to perform CN calling with (single and multiple entries possible, use ',' as delimiter) [ cnvkit, sequenza ]

Optional input:
    * output: the folder where to publish output (default: output)
    * VROOM_CONNECTION_SIZE: value for the environment variable VROOM_CONNECTION_SIZE which sometimes causes trouble with sequenza (default: 500000000)
    * cpus: the number of CPUs used by each job (default: 1)
    * memory: the amount of memory used by each job (default: 4g)

Output:
    CNVkit:  
    * cnvkit/*.antitarget.bed : antitarget regions  
    * cnvkit/*.target.bed : target regions  
    * cnvkit/reference.cnn  
    * cnvkit/*.tumor.targetcoverage.cnn : binned tumor coverage in target region  
    * cnvkit/*.normal.targetcoverage.cnn : binned normal coverage in target region  
    * cnvkit/*.tumor.antitargetcoverage.cnn : binned tumor coverage in anti-target region  
    * cnvkit/*.normal.antitargetcoverage.cnn : binned normal coverage in ani-target region  
    * cnvkit/*.tumor.bintest.cns  
    * cnvkit/*.tumor.cnr : copy number ratios  
    * cnvkit/*.tumor.cns  
    * cnvkit/*.tumor.call.cns : copy number segments  
    Sequenza:  
    * sequenza/*.gz
    * sequenza/*.binned.gz  
    * sequenza/*_alternative_solutions.txt  
    * sequenza/*_confints_CP.txt  
    * sequenza/*_segments.txt  
    * sequenza/*_sequenza_log.txt : sequenza log file  
    * sequenza/*_alternative_fit.pdf  
    * sequenza/*_chromosome_depths.pdf  
    * sequenza/*_chromosome_view.pdf  
    * sequenza/*_CN_bars.pdf  
    * sequenza/*_CP_contours.pdf
    * sequenza/*_gc_plots.pdf  
    * sequenza/*_genome_view.pdf
    * sequenza/*_model_fit.pdf  
    * sequenza/*_sequenza_cp_table.RData  
    * sequenza/*_sequenza_extract.RData

Pipeline output

More details will be provided soon...

Current limitations

  • So far, there are no nf-core modules for Sequenza processes SEQUENZAUTILS_SEQZBINNING and SEQUENZA_R. Therefore, these steps are implemented locally.

Roadmap

Features that will be implemented in the future are listed here:

  • Add ascat as CNV calling tool
  • Add FACETS as CNV calling tool
  • Add TITAN as CNV calling tool

Authors & Acknowledgements

The TronFlow Copy Number Calling pipeline was originally developed by Pablo Riesgo-Ferreiro at TRON - Translational Oncology at the Medical Center of the Johannes Gutenberg University Mainz gGmbH (non-profit). Julian T. Mohr, also at TRON, later joined the project and helped with further development and integration of additional CNV calling tools. Furthermore, Jonas Ibn-Salem and Matthias Peter, also at TRON, supported the project.

Maintenance is now lead by Pablo Riesgo-Ferreiro and Julian T. Mohr.

Main developers:

We thank the following people for their assistance and support in the development of this pipeline:

  • Jonas Ibn-Salem
  • Matthias Peter

Contributing & Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Please report issues using the issue tracker of GitHub.

Citations

If you use TronFlow Copy Number Calling for your analysis, please cite the Zenodo record for a specific version using the following doi: 10.5281/zenodo.7248131

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

CHANGELOG