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Illumina Infectious Disease Software
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  • 👾Illumina Infectious Disease and Microbiology Software
  • DRAGEN Microbial Amplicon
    • ▶️DRAGEN Microbial Amplicon App Documentation
      • 🌀How to start
      • Page
      • 🧬Custom reference
        • 📄Reference BED file format
        • 📄PCR Primer definition file formats
      • 📂Output files
      • 📖Understanding the BaseSpace Reports
        • 📄Summary
        • 📄Sample Report
      • 💠Pipeline Logic
      • ⭐Special considerations for amplicon detection
      • ❓Frequently Asked Questions (FAQ)
  • DRAGEN Targeted Microbial
    • ▶️DRAGEN Targeted Microbial App Documentation
      • 🌀How to set up and run an analysis
      • 🧬Custom genomes and primer sets
        • 📄Genome definition file formats
        • 📄Primer definition file formats
      • ⚙️App Settings
      • 📖Understanding the BaseSpace Reports
        • 📄Summary Report
        • 📄Result Reports
      • 📂Output files
      • 💠Pipeline Logic
      • ⭐Special considerations for amplicon sequencing with IMAP protocols
      • ❓Frequently Asked Questions (FAQ)
      • 🚩Known issues
  • DRAGEN Microbial Enrichment Plus
    • ▶️DRAGEN Microbial Enrichment Plus App Documentation
      • 🌀How to set up and run an analysis
        • 🧬Custom reference FASTA and BED files
        • 📄Microorganism Reporting File format
      • 📂Output files
        • 📖Understanding the BaseSpace HTML reports
        • 📖Report JSON format
      • 💠Pipeline logic
      • ⭐Test information
        • 📄RPIP
        • 📄UPIP
        • 📄RVOP/RVEK
        • 📄VSP
        • 📄VSP V2
        • 📄Custom Panel
      • 🕵️‍♀️Scientific evidence
      • ❓Frequently Asked Questions (FAQ)
      • 🚩Release notes
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  • Overview
  • Input
  • Pipeline steps
  • Output
  • Currently supported platforms
  • Important Notes

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  1. DRAGEN Microbial Amplicon

DRAGEN Microbial Amplicon App Documentation

PreviousIllumina Infectious Disease and Microbiology SoftwareNextHow to start

Last updated 5 months ago

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Overview

DRAGEN Microbial Amplicon is a software application designed to analyze sequencing data from amplicon library preps (both DNA and RNA) on microbiological samples, with an emphasis on viruses. Illumina sequencing reads are processed to generate 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.

Input

Data can be provided in one of the following ways:

  • 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 amplicon primer schemes

  • Chikungunya

    • Illumina

  • Dengue

    • Serotype 1 - Illumina

    • All serotypes -

  • Influenza / - Universal

  • Mpox

    • Pan-clade -

    • Clade I - Illumina

    • Clade II -

  • RSV

  • SARS-CoV-2 - ARTIC

  • Zika -

Custom genome and primer sets

Users can upload custom files to provide user-defined reference genome set and primer definitions. Multiplexed amplicon panels targeting multiple organisms in the same reaction are supported.

Pipeline steps

  1. Align reads to the default reference genome or selected reference genomes using DRAGEN v4.3.6

  2. Trim primer sequences in aligned reads based on coordinates

  3. Filter out samples with insufficient amplicon coverage

  4. Call sequence variants from the alignments using DRAGEN Somatic v4.3.6 and apply them to the corresponding reference genomes to create consensus sequences

  5. If applicable, run Nextclade/Pangolin on the consensus sequences

Output

  • Consensus sequences representing a best estimate of targeted sequences

  • Tables and plots reporting read counts, coverage, and Nextclade/Pangolin results

Currently supported platforms

  • BaseSpace Sequence Hub

Important Notes

  • The sequences are labeled according to the best match in the reference database, which is 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.

Trim and filter reads using

Remove off-target reads using DRAGEN v4.3.6 kmer classifier (for custom reference, remove human reads using a modified version of the v2.2.1)

For organisms with one default reference genome, skip this step. For organisms with multiple candidates, trim primer sequences in reads using , perform assembly using , cluster contigs using , map contigs to candidate reference genomes using , then select reference genomes based on the mapping

▶️
🧬Custom reference
Trimmomatic
SRA Human Read Scrubber tool
Trimmomatic
MEGAHIT
CD-HIT-EST
minimap2
💠Pipeline Logic
📂Output files
Grubaugh Lab
DengueSeq from Grubaugh Lab
A
B
ARTIC
Grubaugh Lab
CDC
WCCRRI
v5.4.2
v5.3.2, v4.1, v4, v3
Grubaugh Lab