🌀How to set up and run an analysis

1. Launch the DRAGEN Targeted Microbial BaseSpace application.

2. After choosing a name and destination project for the Analysis, choose either “Biosample” or “Project” as input type. Selecting “Project” will result in all biosamples in the selected project being analyzed.

3. Next, choose between Enrichment and Amplicon for Experiment Type. Libraries prepared with IMAP should be run as “Amplicon” experiments. Choose the appropriate Amplicon Primer Set that matches the primer design used to prepare your samples or choose “Custom” to provide your own genome references and primer designs. Note that all provided files must first be uploaded to a BaseSpace project before they can be selected in the software.

4. To use a custom reference and primer design, click the “Custom Reference” block to expand it.

5. At a minimum, the user must provide a custom genome reference containing one or more target sequences (to be used for alignment, variant calling and consensus generation) in the form of a FASTA file.

   1. Optionally, the user may provide a BED file that assigns human-readable names and segment numbers (if applicable) to each sequence in the provided FASTA file. Note that the accessions in the genome definition file must match the first part (before whitespace) of the FASTA headers. See the pages for “Genome Definition File Format Specification” in the “Supporting Information” section of this user guide for information on the required format of this file.

   2. Optionally, the user may provide a file containing the locations or sequences of the primers used to prepare this sample. These primer definitions are important to guide the trimming of primer sequence from reads that overlap the binding sites, as well as to define the boundaries of the amplicons whose coverage is used to determine if the sample has sufficient viral material to reliably call variants and generate consensus sequence.

   3. Optionally, the user may choose one or more NextClade datasets to use for phylogenetic analysis of the consensus sequences generated from the samples.

6. Check the appropriate boxes to enable or disable Pangolin and/or NextClade analysis if desired. These tools perform phylogenetic analysis and lineage assignment for SARS-CoV-2 (Pangolin) and other viruses (NextClade). Depending on the chosen Amplicon primer set, not all of these options may be applicable.

7. Click “Launch Application” to begin the Analysis.