We have a new bioinformatic resource that largely replaces the functionality of this project! See our new repository here: https://github.com/epi2me-labs/wf-human-sv

This repository is now unsupported and we do not recommend its use. Please contact Oxford Nanopore: [email protected] for help with your application if it is not possible to upgrade to our new resources, or we are missing key features.

pipeline-structural-variation

Overview

pipeline-structural-variation is a pipeline for calling structural variations on whole genome sequencing data obtained from Oxford Nanopore sequencing platforms. It accepts FASTQ files and outputs aligned reads and filtered SV calls.

Features

The pipeline performs the following steps:

• Maps reads using lra
• Produces QC report using NanoPlot [optional]
• Estimates appropriate parameters for variant calling depending on read depth
• Calls variants using cuteSV
• Filters variants by minimum/maximum length, read support, or type (e.g. insertion, deletion, etc.)

Getting Started

Requirements

The following software packages must be installed prior to running:

• miniconda3 - please refer to installation instructions.

Installation

After installing miniconda3 (see above), install the pipeline as follows:

# Get pipeline
$ wget -O pipeline-structural-variation.tar.gz https://github.com/nanoporetech/pipeline-structural-variation/archive/v2.0.2.tar.gz
$ tar xvzf pipeline-structural-variation.tar.gz
# Change to directory
$ cd pipeline-structural-variation*
# Create conda environment with all dependencies
$ conda env create -n pipeline-structural-variation-v2 -f env.yml
# Activate environment
$ conda activate pipeline-structural-variation-v2
# To test if the installation was successful run
$ snakemake -p -j 1 --configfile config.yml
# Deactivate environment
$ conda deactivate

Input

To run the pipeline the following input files are required:

Output

The main output files created by the pipeline are:

After the pipeline has finished you can find the aligned reads in {sample_name}/alignment/ and the indexed and zipped VCF file in {sample_name}/sv_calls/{sample_name}_cutesv_filtered.vcf.gz, where {sample_name} is derived from the snakemake configuration (see below).

Usage:

To run the pipeline with default settings invoke snakemake as follows.

$ snakemake all --config input_fastq=/data/pass/ reference_fasta=/data/ref/hg38.fa threads=30

all is the default target (see Targets); this will run all steps required for SV calling and produce a QC report for the input reads using NanoPlot. input_fastq specifies the input FASTQ files or a folder containing multiple input FASTQ files (e.g. the pass folder from MinKNOW).

Targets

Options

The pipeline accepts several input parameters. They can either be changed in the config.yml (see below) file or specified when running snakemake.

For example:

snakemake eval --config input_fastq=/data/pass/ reference_fasta=/data/ref/hg38.fa min_sv_length=100 threads=30

In the above example the minimum SV length parameter was changed to 100 bp.

Required parameters

These parameters have to be specified to run the pipeline.

Optional parameters

Annotating and visualising the results

Annotate

This step will become part of the pipeline in the future. Currently, use tools like bedtools or vcfanno to annotate the VCF file with information about genes, repeats, known SV etc. from GTF/GFF/BED files.

There are many ways to retrieve annotations. A few examples are:

• UCSC genome browser
• ENSEMBL
• GENCODE

Visualise

Integrated genome viewer

Download from here.

Supported formats: BAM, BED, VCF, WIG, …

Recommended settings for SV analysis:

View -> Preferences -> Alignments

• Quick consensus mode on
• Hide indels < 10 bp
• Label indels > 30 bp
• Mapping quality threshold: 20

Works well for:

• Deletions and insertions
• Inspect reference sequence and flanking regions
• Compare to annotations
• Inspect heterozygous variants

Ribbon

Find here. Supported format: BAM, BED, VCF

Recommended settings for SV analysis:

• Multi-read settings -> minimum number of alignments -> 4-5
• Single-read settings -> Dot plot

Works well for:

• Visualizing split alignments
• Inversions, translocations, duplications

Results

We benchmarked the pipeline against the preliminary Genome in a bottle SV truth set for HG002.

Precision and Recall

The pipeline was run using auto for determining the most suitable cuteSV parameters to get a good balance between precision and recall. Depending on your application you might want to change the min_read_support parameter to maximize either precision or recall.

* Coverage was computed using mosdepth from the BAM file. The BAM file was neither filtered by mapping quality nor read length.

Reproducing the results

Run the pipeline with HG002 data using the eval target. In the output folder you will find eval/summary.txt containing precision and recall values.

Help

Licence and Copyright

(c) 2020 Oxford Nanopore Technologies Ltd.

This Source Code Form is subject to the terms of the Mozilla Public License, v. 2.0. If a copy of the MPL was not distributed with this file, You can obtain one at http://mozilla.org/MPL/2.0/.

FAQs

I only want my results! What should I do? Install pipeline (see Installation) and run as follows.

$ snakemake all --config input_fastq=/data/pass/ reference_fasta=/data/ref/hg38.fa threads=16

You will find your results in the following locations:

Aligned reads:

sv_sample01/alignment/my_sample_lra.bam

Variant calls:

sv_sample01/sv_calls/my_sample_cutesv_filtered.vcf.gz

What kind of structural variants are supported?

Currently the pipeline has been validated to detect insertions, deletions and duplications (currently reported as insertions as in the GIAB truthset) from whole genome sequencing data. Support for inversions and translocations will be added in the future.

What is the minimum SV length supported?

Currently, 30 bp.

How can i filter my reads by q-score?

The most recent version of MinKNOW will perform filtering automatically. When using the FASTQ files from the pass output folder no additional filtering is required. For unfiltered datasets use NanoFilt with a minimum q-score of 6.

How long will it take to run the pipeline for a 30X human dataset?

When running with 30 CPU cores this will take roughly 3-4 hours.

How much memory will I need?

Memory consumption is determined by lra. Therefore, 16 GB will be required for human datasets. For smaller genomes 8 GB will be sufficient.

How much storage will I need?

Unzipped FASTQ files for a human 30X human dataset will require roughly 200 GB. In addition 150 GB will be required for the mapped BAM files.

Are FAST5 files required to run the pipeline?

No.

Can I use the pipeline to detect variants in a cancer dataset or to detect somatic variants?

The pipeline has not yet been validated on cancer samples or for somatic variant detection.

Can I use the pipeline to detect SVs for targeted sequencing experiments (e.g. long amplicons, etc.)?

Calling variants in targeted regions is supported but requires the user to specify a BED file containing the coordinates of the targeted regions.

For example, save the following in a file called targets.bed

chr1 6579767 6589767

and run pipeline as follows:

snakemake eval --config input_fastq=/data/pass/ reference_fasta=/data/ref/hg38.fa target_bed=targets.bed threads=30

Make sure that the chromosome names in the BED file match the names in your reference FASTA files (e.g. chr1 vs. 1).

Can I use this pipeline to detect gene fusions using DNA data?

The current version does not support calling gene fusions from DNA data as translocation calling is not supported yet. However, support for translocations will be available in the future.

Can I use this pipeline to detect gene fusion using cDNA data?

cDNA data is not supported.

Abbreviations and glossary

References and Supporting Information

If you use this pipeline please cite:

• Ren L, Chaisson M (2020). lra: the Long Read Aligner for Sequences and Contigs. BioRxiv doi: https://doi.org/10.1101/2020.11.15.383273
• Jiang, T., Liu, Y., Jiang, Y. et al. (2020).Long-read-based human genomic structural variation detection with cuteSV. Genome Biol 21, 189. https://doi.org/10.1186/s13059-020-02107-y
• Pedersen, B. S., & Quinlan, A. R. (2018). Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics, 34(5), 867–868. https://doi.org/10.1093/bioinformatics/btx699

When using the QC report please also cite:

• De Coster, W., D’Hert, S., Schultz, D. T., Cruts, M., & Van Broeckhoven, C. (2018). NanoPack: visualizing and processing long-read sequencing data. Bioinformatics, 34(15), 2666–2669. https://doi.org/10.1093/bioinformatics/bty149

For additional information on SV and SV calling please see:

• Nanopore Structural Variation Knowledge exchange
• 100 tomato genomes in 100 days

Research Release

Research releases are provided as technology demonstrators to provide early access to features or stimulate Community development of tools. Support for this software will be minimal and is only provided directly by the developers. Feature requests, improvements, and discussions are welcome and can be implemented by forking and pull requests. However much as we would like to rectify every issue and piece of feedback users may have, the developers may have limited resource for support of this software. Research releases may be unstable and subject to rapid iteration by Oxford Nanopore Technologies.