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The Read-based Taxonomy Classification Workflow

Workflow Overview

The pipeline takes in sequencing files (single- or paired-end) and profiles them using multiple taxonomic classification tools with the Cromwell as the workflow manager.

Workflow Availability

The workflow is available in GitHub: https://github.com/microbiomedata/ReadbasedAnalysis; the corresponding Docker image is available in DockerHub:

Requirements for Execution:

(recommendations are in bold)

  • WDL-capable Workflow Execution Tool (Cromwell)
  • Container Runtime that can load Docker images (Docker v2.1.0.3 or higher)

Hardware Requirements:

  • Disk space: 152 GB for databases (55 GB, 89 GB, and 8 GB for GOTTCHA2, Kraken2 and Centrifuge databases, respectively)
  • 60 GB RAM

Workflow Dependencies

Third party software:

(These are included in the Docker image.)

Requisite databases:

The database for each tool must be downloaded and installed. These databases total 152 GB.

  • GOTTCHA2 database (gottcha2/):

The database RefSeqr90.cg.BacteriaArchaeaViruses.species.fna contains complete genomes of bacteria, archaea and viruses from RefSeq Release 90. The following commands will download the database:

wget https://edge-dl.lanl.gov/GOTTCHA2/RefSeq-r90.cg.BacteriaArchaeaViruses.species.tar
tar -xvf RefSeq-r90.cg.BacteriaArchaeaViruses.species.tar
rm RefSeq-r90.cg.BacteriaArchaeaViruses.species.tar
  • Kraken2 database (kraken2/):

This is a standard Kraken 2 database, built from NCBI RefSeq genomes. The following commands will download the database:

mkdir kraken2
wget https://genome-idx.s3.amazonaws.com/kraken/k2_standard_20201202.tar.gz
tar -xzvf k2_standard_20201202.tar.gz -C kraken2
rm k2_standard_20201202.tar.gz
  • Centrifuge database (centrifuge/):

This is a compressed database built from RefSeq genomes of Bacteria and Archaea. The following commands will download the database:

mkdir centrifuge
wget https://genome-idx.s3.amazonaws.com/centrifuge/p_compressed_2018_4_15.tar.gz
tar -xzvf p_compressed_2018_4_15.tar.gz -C centrifuge
rm p_compressed_2018_4_15.tar.gz

Sample dataset(s):

  • Soil microbial communities from the East River watershed near Crested Butte, Colorado, United States - ER_DNA_379 metagenome (SRR8553641) with metadata available in the NMDC Data Portal. This dataset has 18.3G bases
    • The zipped raw fastq file is available here
  • Zymobiomics mock-community DNA control (SRR7877884); this dataset is has 6.7G bases.
    • The non-interleaved raw fastq files are available as R1 and R2
    • The interleaved raw fastq file is available here
    • A 10% subset of the interleaved fastq is available here

Input:

A JSON file containing the following information:

  1. selection of profiling tools (set as true if selected)
  2. the paths to the required database(s) for the tools selected
  3. the paths to the input fastq file(s) (paired-end data is shown; this can be the output of the Reads QC workflow in interleaved format which will be treated as single-end data.)
  4. the prefix for the output file names
  5. the path of the output directory
  6. CPU number requested for the run.
{
    "ReadbasedAnalysis.enabled_tools": {
        "gottcha2": true,
        "kraken2": true,
        "centrifuge": true
    },
    "ReadbasedAnalysis.db": {
        "gottcha2": "/path/to/database/RefSeq-r90.cg.BacteriaArchaeaViruses.species.fna",
        "kraken2": " /path/to/kraken2",
        "centrifuge": "/path/to/centrifuge/p_compressed"
    },
    "ReadbasedAnalysis.reads": [
        "/path/to/SRR7877884.1.fastq.gz",
        "/path/to/SRR7877884.2.fastq.gz"
    ],
    "ReadbasedAnalysis.paired": true,
    "ReadbasedAnalysis.prefix": "SRR7877884",
    "ReadbasedAnalysis.outdir": "/path/to/ReadbasedAnalysis",
    "ReadbasedAnalysis.cpu": 4
}

Output:

The workflow creates an output JSON file and individual output sub-directories for each tool which include tabular classification results, a tabular report, and a Krona plot (html).

ReadbasedAnalysis/
|-- SRR7877884.json
|-- centrifuge
|   |-- SRR7877884.classification.tsv
|   |-- SRR7877884.report.tsv
|   `-- SRR7877884.krona.html
|
|-- gottcha2
|   |-- SRR7877884.full.tsv
|   |-- SRR7877884.krona.html
|   `-- SRR7877884.tsv
|
`-- kraken2
    |-- SRR7877884.classification.tsv
    |-- SRR7877884.krona.html
    `-- SRR7877884.report.tsv

Below is an example of the output directory files with descriptions to the right.

Directory/File Name Description
SRR7877884.json ReadbasedAnalysis result JSON file
centrifuge/SRR7877884.classification.tsv Centrifuge output read classification TSV file
centrifuge/SRR7877884.report.tsv Centrifuge output report TSV file
centrifuge/SRR7877884.krona.html Centrifuge krona plot HTML file
gottcha2/SRR7877884.full.tsv GOTTCHA2 detail output TSV file
gottcha2/SRR7877884.tsv GOTTCHA2 output report TSV file
gottcha2/SRR7877884.krona.html GOTTCHA2 krona plot HTML file
kraken2/SRR7877884.classification.tsv Kraken2 output read classification TSV file
kraken2/SRR7877884.report.tsv Kraken2 output report TSV file
kraken2/SRR7877884.krona.html Kraken2 krona plot HTML file

Version History

  • 1.0.8 (release date 07/23/2024)
  • 1.0.1 (release date 01/14/2021; previous versions: 1.0.0)

Point of contact