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DOI Continuous Integration

Hutspot

This is a multi sample DNA variant calling pipeline based on Snakemake, bwa and the GATK HaplotypeCaller.

Features

  • Any number of samples is supported
  • Whole-genome calling, regardless of wet-lab library preparation.
  • Follows modern best practices
    • Each sample is individually called as as a GVCF.
    • A VCF is then produced by genotyping the individual GVCFs separately for each sample.
  • Data parallelization for calling and genotyping steps.
    • Using the scatter_size setting in the configuration file, the reference genome is split into chunks, and each chunk can be processed independenly. The default value of 1 billon will scatter the human reference genoom into 6 chunks.
  • Reasonably fast.
    • 96 exomes in < 24 hours.
  • No unnecessary jobs
  • Calculate coverage metrics if a bedfile is specified.
  • Fully containerized rules through singularity and biocontainers. Legacy conda environments are no long available.

Installation

This repository contains a conda environment file that you can use to install all dependencies in a conda environment:

conda env create -f environment.yml

Singularity

We highly recommend the user of the containerized rules through singularity.

This option does require you to install singularity on your system. As this usually requires administrative privileges, singularity is not contained within our provided conda environment file.

If you want to use singularity, make sure you install version 3 or higher.

Debian

If you happen to use Debian buster, singularity 3.0.3 comes straight out of the box with a simple:

sudo apt install singularity-container

Docker

You can run singularity within a docker container. Please note that the container MUST run in privileged mode for this to work.

We have provided our own container that includes singularity and snakemake here.

Manual install

If you don't use Debian buster and cannot run a privileged docker container, you - unfortunately :-( - will have to install singularity manually. Please see the installation instructions here on how to do that.

Operating system

Hutspot was tested on Ubuntu 16.04 only. It should reasonably work on most modern Linux distributions.

Requirements

For every sample you wish to analyze, we require one or more paired end readgroups in fastq format. They must be compressed with either gzip or bgzip.

The configuration must be passed to the pipeline through a configuration file. This is a json file listing the samples and their associated readgroups as well as the other settings to be used. An example config json can be found here, and a json schema describing the configuration file can be found here. This json schema can also be used to validate your configuration file.

Reference files

The following reference files must be provided in the configuration:

  1. reference: A reference genome, in fasta format. Must be indexed with samtools faidx.
  2. dbsnp: A dbSNP VCF file
  3. known_sites: One ore more VCF files with known sites for base recalibration

The following reference files may be provided:

  1. targetsfile: Bed file of the targets of the capture kit. Used to calculate coverage.
  2. baitsfile: Bed file of the baits of the capture kit. Used to calculate picard HsMetric.
  3. refflat: A refFlat file to calculate coverage over transcripts.
  4. scatter_size: Size of the chunks to split the variant calling into.
  5. female_threshold: Fraction of reads between X and the autosomes to call as female.

How to run

After installing and activating the main conda environment, as described above, the pipeline can be started with:

snakemake -s Snakefile \
--use-singularity \
--configfile tests/data/config/sample_config.json

This would start all jobs locally. Obviously this is not what one would regularly do for a normal pipeline run. How to submit jobs on a cluster is described later. Let's first move on to the necessary configuration values.

Configuration values

The required and optional outputs are specified in the json schema located in config/schema.json. Before running, the content of the --configfile is validated against this schema.

The following configuration values are required:

configuration description
reference Absolute path to fasta file
samples One or more samples, with associated fastq files
dbsnp Path to dbSNP VCF file
known_sites Path to one or more VCF files with known sites. Can be the same as the dbsnp file

The following configuration options are optional:

configuration description
targetsfile Bed file of the targets of the capture kit. Used to calculate coverage
baitsfile Bed file of the baits of the capture kit. Used to calculate picard HsMetrics
female_threshold Float between 0 and 1 that signifies the threshold of the ratio between coverage on X/overall coverage that 'calls' a sample as female. Default = 0.6
scatter_size The size of chunks to divide the reference into for parallel execution. Default = 1000000000
coverage_threshold One or more threshold coverage values. For each value, a sample specific bed file will be created that contains the regions where the coverage is above the threshold
restrict_BQSR Restrict GATK BaseRecalibration to a single chromosome. This is faster, but the recalibration is possibly less reliable
multisample_vcf Create a true multisample VCF file, in addition to the regular per-sample VCF files

Cluster configuration

To run on a cluster, snakemake needs to be called with some extra arguments. Additionally, it needs a cluster yaml file describing resources per job.

If you run on a cluster with drmaa support,an environment variable named DRMAA_LIBRARY_PATH must be in the executing shell environment. This variable points to the .so file of the DRMAA library.

An sge-cluster.yml is bundled with this pipeline in the cluster directory. It is optimized for SGE clusters, where the default vmem limit is 4G. If you run SLURM, or any other cluster system, you will have to write your own cluster yaml file. Please see the snakemake documentation for details on how to do so. Given the provided sge-cluster.yml, activating the cluster mode can be done as follows:

snakemake -s Snakefile \
--cluster-config cluster/sge-cluster.yml
--drmaa ' -pe <PE_NAME> {cluster.threads} -q all.q -l h_vmem={cluster.vmem} -cwd -V -N hutspot' \

Limitations

Sample names should be unique, and not overlap (such as sample1 and sample10). This is due to the way output files are parsed by multiQC, when sample names overlap, the json output for picard DuplicationMetrics cannot be parsed unambiguously.

Binding additional directories under singularity

In singularity mode, snakemake binds the location of itself in the container. The current working directory is also visible directly in the container.

In many cases, this is not enough, and will result in FileNotFoundErrors. E.g., suppose you run your pipeline in /runs, but your fastq files live in /fastq and your reference genome lives in /genomes. We would have to bind /fastq and /genomes in the container.

This can be accomplished with --singularity-args, which accepts a simple string of arguments passed to singularity. E.g. in the above example, we could do:

snakemake -S Snakefile \
--use-singularity  \
--singularity-args ' --bind /fastq:/fastq --bind /genomes:/genomes '

Summing up

To sum up, a full pipeline run under a cluster would be called as:

snakemake -s Snakefile \
--use-singularity \
--singularity-args ' --bind /some_path:/some_path ' \
--cluster-config cluster/sge-cluster.yml \
--drmaa ' -pe <PE_NAME> {cluster.threads} -q all.q -l h_vmem={cluster.vmem} -cwd -V -N hutspot' \
--rerun-incomplete \
--jobs 200 \
-w 120 \
--max-jobs-per-second 30 \
--restart-times 2 \
--configfile config.json

Graph

Below you can see the rule graph of the pipeline. The main variant calling flow is highlighted in red. This only shows dependencies between rules, and not between jobs. The actual job graph is considerably more complex, as nearly all rules are duplicated by sample and some (the scatter jobs) additionally by chunk.

As a rough estimate of the total number of jobs in pipeline you can use the following formula:

This gives about 12,000 jobs for a 96-sample run with 2 bed files and 100 chunks.

NOTE: the graph will only render if your markdown viewer supports plantuml. Having trouble viewing the graph? See this static SVG instead.

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	22[label = "covstats", color = "0.39 0.6 0.85", style="rounded"];
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	24[label = "create_markdup_tmp", color = "0.41 0.6 0.85", style="rounded,dashed"];
	25[label = "sickle", color = "0.19 0.6 0.85", style="rounded"];
	26[label = "genome", color = "0.62 0.6 0.85", style="rounded"];
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}

LICENSE

AGPL-3.0