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This pipeline uses centrifuge for microbial classification and enables rapid, accurate, and sensitive labeling of reads and quantification of species.

This pipeline is part of BioWDL developed by the SASC team at Leiden University Medical Center.

Usage

You can run this pipeline using Cromwell:

java \
    -jar cromwell-<version>.jar \
    run \
    -i inputs.json \
    talon-wdl.wdl

Inputs

Inputs are provided through a JSON file. The minimally required inputs are described below, but additional inputs are available. A template containing all possible inputs can be generated using Womtool as described in the WOMtool documentation. For an overview of all available inputs, see this page.

{
    "Classification.sampleConfigFile": "A sample configuration file (see below).",
    "Classification.outputDirectory": "The path to the output directory.",
    "Classification.dockerImagesFile": "A file listing the used docker images.",
    "Classification.sampleWorkflow.centrifugeIndex": "The files of the centrifuge index for the reference genomes."
}

Sample configuration

Verification

All samplesheet formats can be verified using biowdl-input-converter. It can be installed with pip install biowdl-input-converter or conda install biowdl-input-converter (from the bioconda channel). Python 3.7 or higher is required.

With biowdl-input-converter --validate samplesheet.csv The file “samplesheet.csv” will be checked. Also the presence of all files in the samplesheet will be checked to ensure no typos were made. For more information check out the biowdl-input-converter readthedocs page.

CSV format

The sample configuration can be given as a csv file with the following columns: sample, library, readgroup, R1, R1_md5, R2, R2_md5.

column name	function
sample	sample ID
library	library ID. These are the libraries that are sequenced. Usually there is only one library per sample.
readgroup	readgroup ID. Usually a library is sequenced on multiple lanes in the sequencer, which gives multiple fastq files (referred to as readgroups). Each readgroup pair should have an ID.
R1	The fastq file containing the first reads of the read pairs.
R1_md5	Optional: md5sum for the R1 file.
R2	Optional: The fastq file containing the reverse reads.
R2_md5	Optional: md5sum for the R2 file.

The easiest way to create a samplesheet is to use a spreadsheet program such as LibreOffice Calc or Microsoft Excel, and create a table:

sample	library	read	R1	R1_md5	R2	R2_md5
Sample1	lib1	rg1	tests/data/norovirus.paired.end.1.R1.fastq.gz	1ff9f164f633933046b725ed7116354c	tests/data/norovirus.pairedEnd.1.R2.fastq.gz	f18ae76ff14b557ea4b0fc6a787b5d12
Sample2	lib1	rg1	tests/data/norovirus.paired.end.2.R1.fastq.gz	8d8d01381a35787711ee47bf7dde55b0	tests/data/norovirus.pairedEnd.2.R2.fastq.gz	a829f3334994bbd0411dbebe6ac62223

NOTE: R1_md5, R2 and R2_md5 are optional do not have to be filled. And additional fields may be added (eg. for documentation purposes), these will be ignored by the pipeline.

After creating the table in a spreadsheet program it can be saved in csv format.

Example

The following is an example of what an inputs JSON might look like:

{
    "Classification.sampleConfigFile": "tests/samplesheets/paired.end.csv",
    "Classification.outputDirectory": "tests/test-output",
    "Classification.dockerImagesFile": "dockerImages.yml",
    "Classification.sampleWorkflow.centrifugeIndex": [
        "tests/data/index/norovirus.1.cf",
        "tests/data/index/norovirus.2.cf",
        "tests/data/index/norovirus.3.cf",
        "tests/data/index/norovirus.4.cf"
    ]
}

Dependency requirements and tool versions

Biowdl pipelines use docker images to ensure reproducibility. This means that biowdl pipelines will run on any system that has docker installed. Alternatively they can be run with singularity.

For more advanced configuration of docker or singularity please check the cromwell documentation on containers.

Images from biocontainers are preferred for biowdl pipelines. The list of default images for this pipeline can be found in the default for the dockerImages input.

Output

The workflow will output trimmed reads from the QC pipeline, a centrifuge classification file, a alignment metrics file, a report file per sample and a krona plot per sample.

Contact

For any questions about running this pipeline and feature request (such as adding additional tools and options), please use the github issue tracker or contact the SASC team directly at: sasc@lumc.nl.