GMSMetaPost

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This repository was archived on 2024-04-18 as it was no longer in use or being developed

Overview of the pipeline

Please note that this is not the finalized overview of the pipeline and maybe subject to changes.

GMSMetaPost takes the output files of three classifiers from taxprofiler (kraken2, centrifuge, kaiju).

• Calculates reads per million (RPM) for each classifier.
• Concatenates the different tsv files from each classifier into one.
• Filters RPM based on a threshold given by the user.
• Validate and remove missing taxonomic IDs.
• Download potential reference genomes from a local BlAST nt database (See below on how to install the local blast db).
• Coverage plots for potential significant findings.

Prerequisites

Metadata files

For running the pipeline two metadata files are required: samples.csv and samplesheet.csv. Here are examples of how they may look like:

• samples.csv

sample,run_accession,instrument_platform,fastq_1,fastq_2,fasta
SRR12875558_se-SRR12875558,SRR12875558,OXFORD_NANOPORE,assets/input/se/SRR12875558_1.fastq.gz,,
SRR12875570_pe-SRR12875570,SRR12875570,ILLUMINA,assets/input/pe/SRR12875570_1.fastq.gz,assets/input/pe/SRR12875570_2.fastq.gz,

• samplesheet.csv

sample,kraken2,centrifuge,kaiju
SRR12875570_pe-SRR12875570,assets/tsvs/SRR12875570_pe-SRR12875570-kraken2.kraken2.report.tsv,assets/tsvs/SRR12875570_pe-SRR12875570-centrifuge.report.tsv,assets/tsvs/SRR12875570_pe-SRR12875570-kaiju.tsv
SRR12875558_se-SRR12875558,assets/tsvs/SRR12875558_se-SRR12875558-kraken2.kraken2.report.tsv,assets/tsvs/SRR12875558_se-SRR12875558-centrifuge.report.tsv,assets/tsvs/SRR12875558_se-SRR12875558-kaiju.tsv

The path to samples.csv must be defined in fastq_data parameter in nextflow.config and path to samplesheet.csv must be defined in input parameter.

Temporary metadata files containing joined taxonomic profiling

At the current stage of this pipeline additional metadata files containing joined taxonomic profiling files are required. They should be located in the root of the directory defined by assets parameter in nextflow.config file. Their names (without the .tsv suffix) must be identical to the sample names given in the two previously mentioned metadata files. E.g. if assets parameter is assets and one of the samples in samples.csv or samplesheet.csv is named SRR12875558_se-SRR12875558 there must be a joined taxonomic profiling file named assets/SRR12875558_se-SRR12875558.tsv

These temporary metadata files can look for example the following:

taxon_name	rpm	taxid	taxonomic_rank	classifier	centrifuge_genome_size	centrifuge_num_reads	centrifuge_abundance	kaiju_percent	kraken2_percentage_fragments_covered	kraken2_num_fragments_covered	reads_count	cami_taxid	cami_rank	cami_taxpath	cami_taxpathsn	cami_percentage
Ungulate tetraparvovirus 3	70062.2	1511916	species	centrifuge	5444	1389.0	0.0				1542
Parvovirus YX-2010/CHN	45663.1	754189	leaf	centrifuge	5444	851.0	0.0				1005

BLAST nt database

GMSMetaPost requires a local version of BLAST nt database. The easiest way to aqcuire it is by downloading with blast v.2.13.0 utility update_blastdb.pl:

update_blastdb.pl \
--decompress \
nt

In addition the latest taxonomy database is required as well. It should be downloaded into the same directory:

wget --continue https://ftp.ncbi.nlm.nih.gov/blast/db/taxdb.tar.gz
rm taxdb.btd taxdb.bti
tar xvf taxdb.tar.gz
rm -f taxdb.tar.gz

It is also recommended to update regularly the local BLAST nt database with command:

update_blastdb.pl \
nt

Finally, the user should define the path to the database by either defining blast_db parameter in nextflow.config or on the command line.

Running GMSMetaPost

GMSMetaPost can be run with command:

nextflow run main.nf \
--input assets/samplesheet.csv \
--outdir test \
-profile conda

conda profile can also be replaced by singularity or docker.

Troubleshooting

• If you get the following error message: no space left on device, make sure that that you are using:

  singularity.cacheDir = "/path/to/cache"

• If you get the following error message for singularity:

  FATAL: While getting image info: error decoding image: invalid ObjectId in JSON

  You are likely using an older version of singularity. You can update it with the command:

  conda install -c conda-forge singularity

Acknowledgements

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x. In addition, references of tools and data used in this pipeline are as follows: