Metagenome Read-based Taxonomy Classification Workflow

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

• GOTTCHA2
• Kraken2
• Centrifuge
• SingleM

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