▶️DRAGEN Targeted Microbial App Documentation

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NOTE: This application will be obsoleted on May 31, 2025. If using an Illumina Infectious Disease/Micro Enrichment panel workflow, use DRAGEN Microbial Enrichment Plus. If using IMAP, IMAP-FLU or COVID-seq kit please start using the DRAGEN Microbial Amplicon app instead.

▶️ DRAGEN Targeted Microbial App Documentation

Overview

Summary

DRAGEN Targeted Microbial is a software application designed to analyze sequencing data from enrichment and amplicon library preps (both DNA and RNA) on microbiological samples, with an emphasis on viruses. Illumina sequencing reads are processed to remove human-origin sequence, then assembled into consensus sequences that represent a best estimate of the population of viral sequences in each sample. Where appropriate, these consensus sequences are further analyzed by the phylogenetic analysis tools NextClade and/or Pangolin to provide an identification of the clade or lineage of each sequence.

Inputs

• Samples / biosamples with FASTQ datasets (see details in library preparation documents)

• A project containing one or more samples / biosamples with FASTQ datasets

  • all samples / biosamples in the selected project will be analyzed

Supported hybrid-capture enrichment panels

• Viral Surveillance Panel (VSP)

• Pan-Coronavirus Panel (Pan-Cov)

• Respiratory Virus Oligo Panel (RVOP)

Supported amplicon primer schemes

• Chikungunya (Grubaugh lab; Illumina)

• Dengue Serotype 1 (Grubaugh Lab; Illumina)

• Influenza A/B (Zhou et al)

• MPXV (Grubaugh Lab)

• Respiratory Syncytial Virus (RSV) (WCCRRI; CDC)

• SARS-CoV-2 (ARTIC v3, v4, v4.1, v5.3.2)

• Zika (Grubaugh lab)

Custom genomes and panels

Supports uploading FASTA files to use as reference genomes for both enrichment and amplicon panels, as well as custom primer definitions for amplicon panels. Multiplexed amplicon panels targeting multiple organisms in the same reaction are supported.

Analysis Pipeline

1. Reads are trimmed and filtered using Trimmomatic with the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36.

2. Human reads are removed with a modified version of the SRA Human Read Scrubber tool.

3. MEGAHIT is used to perform de novo assembly on the scrubbed reads.

4. CD-HIT-EST is used to cluster similar contigs to reduce redundancy.

5. The resulting contigs are mapped to a set of reference genomes using minimap2.

6. The best matching reference for each contig is selected for short read mapping.

7. The scrubbed reads from step 2 are aligned to the selected reference genomes using DRAGEN v4.2.4

8. Sequence variants are called from the alignments using DRAGEN Somatic Small Variant Caller v4.2.4 and applied to the corresponding reference sequences to create consensus sequences.

9. If applicable, Pangolin and/or Nextclade are run on the consensus sequences.

Outputs

The software generates consensus sequences representing a best estimate of the population of targeted sequences in the sample. NextClade and Pangolin analysis are run on select organisms. See this page for details:

Output files

Important Notes

• The sequences are labeled according to the best match in the panel references. These references are not exhaustive and the labels should not be taken as definitive for strain-typing. If strain typing is needed, the built-in NextClade and/or Pangolin tools can be used for supported organisms. Alternatively, a BLAST or similar search of nucleotide databases may provide a more detailed match.

• Because of sequence homology, it is possible that organisms with very few reads will result in the generation of a sequence not present (false positive). Although the de novo assembly step of this software largely mitigates such instances, sequences with very low horizontal coverage (< 5%) should be treated with caution and are highlighted as "low confidence" in the reports.

Currently supported platforms

• BaseSpace Sequence Hub (native BaseSpace app)