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Metagenome Read-based Taxonomy Classification Workflow

Workflow Diagram

Workflow Overview

This pipeline profiles sequencing files (single- or paired-end, long- or short-read) using modular, selectable taxonomic classification tools. It supports GOTTCHA2, Kraken2, Centrifuge, and SingleM via Cromwell (WDL) and Docker, enabling scalable, reproducible metagenome analysis.

Supported Tools

Flexible selection of one or more tools via workflow input variables. Each profiler must be enabled via JSON, and paths to reference databases are required.

Workflow Availability

  • GitHub Repository
  • Docker Images:
    • microbiomedata/nmdc_taxa_profilers
    • wwood/singlem:0.20.2
    • microbiomedata/bbtools:38.96

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 for GOTTCHA2, 89 GB for Kraken2, and 8 GB for Centrifuge databases). SingleM public marker database is native to the SingleM container.
  • RAM: 60 GB

Workflow Dependencies

Third-party software (included in the Docker image):

  • GOTTCHA2 v2.1.8.5 (License: BSD-3-Clause-LANL)
  • Kraken2 v2.1.2 (License: MIT)
  • Centrifuge v1.0.4 (License: GPL-3)
  • SingleM v0.20.2 (License: GPL-3)

Requisite Databases

You must download and install each tool's database to use that tool (total: 152 GB):

  • GOTTCHA2 database (gottcha2/):
    • RefSeqr90.cg.BacteriaArchaeaViruses.species.fna contains complete genomes of bacteria, archaea and viruses from RefSeq Release 90.
    • Download commands:
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/):
    • Standard Kraken 2 database, built from NCBI RefSeq genomes.
    • Download commands:
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/):
    • Compressed database built from RefSeq genomes of Bacteria and Archaea.
    • Download commands:
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 Datasets

  • Soil microbial communities (East River watershed near Crested Butte, Colorado, US) — ER_DNA_379 metagenome (SRR8553641) with metadata in the NMDC Data Portal. This dataset has 18.3G bases. Zipped raw fastq file is available here.
  • Zymobiomics mock-community DNA control (SRR7877884), dataset has 6.7G bases.
    • Non-interleaved raw fastq files: R1 and R2
    • Interleaved raw fastq file available here
    • 10% subset of interleaved fastq available here

Input

A JSON file containing:

  1. Selection of profiling tools (optional, default only singlem set true)
  2. Paths to the required database(s) for the selected tools
  3. Paths to the input fastq file(s) (paired-end data shown; output of the Reads QC workflow in interleaved format can be treated as single-end.)
  4. Paired end Boolean
  5. The project name
  6. Long reads Boolean
  7. CPU number requested for the run
{
   "ReadbasedAnalysis.enabled_tools": {
     "gottcha2": false,
     "kraken2": false,
     "centrifuge": false,
     "singlem": 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-int.fastq.gz",
   "ReadbasedAnalysis.paired": true,
   "ReadbasedAnalysis.proj": "SRR7877884",
   "ReadbasedAnalysis.long_read": false,
   "ReadbasedAnalysis.cpu": 8
}

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).

Directory/File Name Description
SRR7877884_profiler.info ReadbasedAnalysis profiler info JSON file
SRR7877884_centrifuge_classification.tsv Centrifuge output read classification TSV file
SRR7877884_centrifuge_report.tsv Centrifuge output report TSV file
SRR7877884_centrifuge_krona.html Centrifuge krona plot HTML file
SRR7877884_gottcha2_full.tsv GOTTCHA2 detail output TSV file
SRR7877884_gottcha2_report.tsv GOTTCHA2 output report TSV file
SRR7877884_gottcha2_krona.html GOTTCHA2 krona plot HTML file
SRR7877884_kraken2_classification.tsv Kraken2 output read classification TSV file
SRR7877884_kraken2_report.tsv Kraken2 output report TSV file
SRR7877884_kraken2_krona.html Kraken2 krona plot HTML file
SRR7877884_singlem_classification.tsv SingleM output read classification TSV file
SRR7877884_singlem_report.tsv SingleM output report TSV file
SRR7877884_singlem_krona.html SingleM krona plot HTML file

Version History

  • 1.1.0 (release date 11/23/2025)

Point of Contact

  • Package maintainers: Chienchi Lo, Po-E Li, Valerie Li