Set Up DRAGEN Secondary Analysis

The MiSeq i100 Series allows you to configure secondary analysis using the DRAGEN applications that are installed on the instrument. Before setting up the secondary analysis, make sure you have installed the appropriate application. For more information on installing applications on the MiSeq i100 Series, refer to Applications.

Configure the analysis application as follows.

1. [Optional] Enter a description for the configuration.
2. Select your library prep kit and index adapter kit.

When an Illumina library prep kit is selected, adapter sequences for Read 1 and Read 2 automatically populate and cannot be modified. Override cycles also automatically populate.

3. Configure the options and settings based on the selected application.
• Adapter Read 1
• Adapter Read 2
• Override Cycles
• FASTQ file compression format
• Keep FASTQ files
• Reference Database
• Read QC
• Read count threshold
• Primer Trimming

If Length is selected, then the following options are available.

• Forward Primer Length
• Reverse Primer Length
• Reference Genome
• DNA or RNA
• Targeted regions
• Variant type
• DNA Genotype of Interest
• CNV panel of normals
• DNA Primer Length
• DNA Phase Variant Distance
• Enable DNA structural variant calling
• RNA gene annotation file
• Enable RNA splice variant analysis
• RNA splice variant knowns
• Enable differential expression
• Map/Align output format
• Reference genome
• Variant type
• Variant callers
• Targeted regions
• Somatic baseline file
• CNV panel of normals
• CNV population SNP VCF
• Germline tagging file
• Map/Align output format
• Reference Genome
• Library input volume
• LibraryQC pipeline mode
• Map/Align output format
• Amplicon Primer Set

If Custom is selected, then the following options are available.

• Custom Reference FASTA for Consensus Generation
• Custom Reference BED (optional)
• Custom PCR Primer Definitions (optional)
• Analysis ID
• Run ID
• Enrichment Panel
• Enrichment Panel Microorganism Reporting List
• Read QC
• Report Bacterial AMR Markers only when an associated microorganism is reported
• AMR Only
• Report microorganisms and/or AMR markers that are below threshold
• Read classification sensitivity
• Nextclade
• Quantitative Internal Control (IC)
• Internal Control Concentration
• Sample ID
• Control Type
• Reference Genome
• Enable down-sampling
• Number of fragments to Downsample
• Pipeline Mode
• RNA gene annotation file
• Targeted regions
• Map/Align output format
• Reference Genome
• Sample ID
• Variant callers
• Ploidy
• Map/Align output format
4. Use one of the following options to enter information for the samples used in secondary analysis:
• Enter sample information in a *.csv file by selecting Download template. To import the edited sample template, select Import samples, and then select the CSV file.
• Paste sample IDs and either index plate well positions or i7 and i5 indexes directly from an external file. Before pasting, enter the number of sample rows in the Rows field, and then select +. Sample IDs can contain up to 100 alphanumeric characters, hyphens, and underscores.

Fixed-layout index plates require entries for well position. Indexes that do not have a fixed layout require entries for i7 and i5 indexes. i5 indexes must be entered in the forward orientation.

5. Select Next, and then review the run details.
6. [Optional] Perform any of the following actions:
• Select Add another configuration to add another configuration. You can have a maximum of 12 configurations.
• To edit the run settings or configuration settings, select Edit next to the run or configuration.
• To delete a configuration, select Delete next to the configuration, and then select Yes, delete.
7. To save the run, select one of the following options:
• To edit the run details later, select Save as draft.
• Select Save as planned to finalize the run details and plan for sequencing.
• To export a sample sheet from a run planned on instrument, select the planned run to open, then under Run Review, select Export sample sheet.