DRAGEN Metagenomics Pipeline in BaseSpace general information

The Illumina DRAGEN Metagenomics Pipeline app performs taxonomic classification of reads using the Kraken2 algorithm and a corresponding taxonomic database. The app provides interactive visualizations and raw classification output for per-sample and aggregate analyses.

This version of the DRAGEN Metagenomics Pipeline App 3.7.0 includes the following features:

• DRAGEN Map/Align

• Built-in de-hosting (human host read removal)

• Built-in kraken2 database

  • MiniKraken2 (March 2020)

  • Extended Kraken2 (March 2020)

  • Kraken2 PlusPFP-8 (May 17 2021)

  • Kraken2 Viral (May 17 2021)

  • This app also supports custom Kraken2-compatible database input as a compressed tar archive. Select 'Custom' from the menu and then use the Custom Reference Database control.

• Organism detection report

  • CSV and HTML in-browser formats

• Adjustable detection thresholds

• Integrated QC metrics

• Single sample and aggregate reports

Workflow

1. Select input sample(s).

2. Choose Reference Database. To use a custom kraken database, first upload the database into BaseSpace as .tar.gz file.

3. Choose a reference genome for de-hosting.

4. Choose whether to enable de-hosting (on by default).

5. Configure the Organism Detection Report (on by default).

   • Choose "From text input" to provide a semi-colon-delimited list of organisms identifiers via the input form. The values must be the scientific names and separated by semi-colons.

     • Example:

       • Severe acute respiratory syndrome coronavirus 2;Human coronavirus 229E;Human coronavirus HKU1;Human coronavirus NL63;Human coronavirus OC43;coronavirus;Influenza A;Influenza

   • Choose "From file input" to provide a list of organism identifiers via a text file in BaseSpace. Text file must have a .txt extension and must have one organism identifier per line.

6. Configure the low and high detection thresholds.

7. Optionally set "Preferred number of BioSamples per node". By default, this value is set to 4.

8. Optionally enable sorting (Advanced Settings). This may impact runtime by up to 6X.

9. Optionally, if de-hosting is enabled, modify the Alignment Minimum Score. This must be greater than 23 and less than or equal to the sample read length. The default value of 50 is recommended, unless analyzing samples shorter than that.

10. Configure Remove Output Alignment (Advanced Settings). By default, all de-hosting BAM related files are deleted. Uncheck to preserve host BAM files.

Input Files

• FASTQ

• Custom Kraken2 Reference (.tar.gz)

• Organism Identifiers File (.txt)

Key Output Files

• .microbe-classification-report.tsv

• .microbe-classification.tsv

• .microbe-classification_metrics.csv

• .organism-detection-report.csv

Known Limitations

• Before running the DRAGEN Metagenomics Pipeline app, be aware of the following limitation:

  • Mixture of single-end and paired-end input data is not supported

• This app supports up to 100 million reads per sample. Use the FastQ Toolkit or another method to down-sample if necessary before analysis.

• The Krona chart is generated by Krona and the Classification and Detection Reports by Kraken, resulting in slight differences in organism names.

Known Issues

• Large custom databases may cause memory issues during analysis, leading to failures. A potential workaround is to downsample the input samples or use a smaller reference database

Example Data Set

To view sample data sets and run outputs here, see the public data project here.

For any feedback or questions regarding this article (Illumina Knowledge Article #3688), contact Illumina Technical Support [e-mail].