The Read-based Taxonomy Classification (v1.0.10)

Workflow Overview

The pipeline takes in a Illumina sequencing file (single- or paired-end) or PacBio sequencing file 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:

• microbiomedata/nmdc_taxa_profilers

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.)

• GOTTCHA2 v2.1.8.5 (License: BSD-3-Clause-LANL)

• Kraken2 v2.1.2 (License: MIT)

• Centrifuge v1.0.4 (License: GPL-3)

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):

For best results, using datasets that have already gone through ReadsQC is strongly encouraged.

Short Reads

• 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 file is here

    • ReadsQC Cleaned File

  • A 10% subset of the interleaved file is available as a quick dataset here

    • ReadsQC Cleaned File

Long-Reads:

Zymobiomics synthetic metagenome (SRR13128014) For testing we have subsampled the dataset (~57MB), the original dataset is ~18G of bases.

• ReadsQC Cleaned File

Input:

A JSON file containing the following information:

1. the path to the input fastq file (this can be the output of the Reads QC workflow in interleaved format which will be treated as single-end data.)

2. if the input is paired (boolean)

3. if the input is long_read (boolean)

4. the prefix for the output file names

5. CPU number requested for the run.

6. project id

{
    "ReadbasedAnalysis.input_file": "SRR7877884-int-0.1.fastq.gz"
    "ReadbasedAnalysis.paired": true,
    "ReadbasedAnalysis.long_read": false,
    "ReadbasedAnalysis.prefix": "SRR7877884",
    "ReadbasedAnalysis.cpu": 8
    "ReadbasedAnalysis.proj": "TEST"
}

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

Download the example ReadbasedAnalysis output for the short-reads Illumina run SRR7877884 (10% subset) here.

Download the example ReadbasedAnalysis output for the long-reads PacBio run SRR13128014 here.

Version History

• 1.0.10 (release date 03/04/2025)

• 1.0.8 (release date 07/23/2024; previous versions: 1.0.0)

Point of contact

• Package maintainers: Chienchi Lo <chienchi@lanl.gov>