Set Up DRAGEN Secondary Analysis

The MiSeq i100 Plus System 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 Plus System, 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.

Setting

Description

Adapter Read 1

Adapter sequence for Read 1. If using an Illumina library prep kit, you cannot modify this field.

Adapter Read 2

Adapter sequence for Read 2. If using an Illumina library prep kit, you cannot modify this field.

Override Cycles

Specifies UMI cycles and mask out cycles of a read. The values are automatically populated according to your library prep and index adapter kit information.

FASTQ file compression format

DRAGEN: Save FASTQ files in *.ora format.

gzip: Save FASTQ files in *.gzip format.

Keep FASTQ files

If storing data locally, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.

Setting

Description

Reference Genome

This application can process one genome per analysis.

For instructions on creating and importing custom reference genomes, refer to Reference Builder (Instruments) support site.

Library input volume

The input volume of each normalized library in the sequencing pool. The library input volume must be equal for all libraries in the pool

LibraryQC pipeline mode

The pipeline mode determines the output files generated.

FastQC: Performs quality checks for FASTQ files.

FullPipeline: Performs FastQC, mapping, and alignment.

Map/Align output format

BAM: The BAM file format stores reads aligned to a reference genome in a compressed binary format

CRAM: CRAM is a reference-based compression file format that offers better compression than BAM.

None: Select this option to delete intermediate files upon completion of secondary analysis.

Setting

Description

Reference Genome

For instructions on creating and importing custom reference genomes, refer to Reference Builder (Instruments) support site.

Sample ID

Maximum of 100 characters.

Alphanumeric characters, underscores, and hyphens are allowed.

Variant callers

Somatic variant caller

Ploidy

Ploidy of the reference genome.

Map/Align output format

BAM: The BAM file format stores reads aligned to a reference genome in a compressed binary format

CRAM: CRAM is a reference-based compression file format that offers better compression than BAM.

None: Select this option to delete intermediate files upon completion of secondary analysis.

Setting

Description

Analysis ID

[Optional] Provide a name for the analysis. When not set, the Analysis ID is used for the time stamp.

Run ID

[Optional] Provide a sequencing run ID. When not set, the system defaults to not applicable.

Enrichment Panel

Select an infectious disease or microbiology enrichment panel from the drop-down menu.

Enrichment Panel Microorganism Reporting List

[Optional] Select report filter setting to define microorganisms listed in the report. Some enrichment panels have pre-defined microorganism reporting lists. Custom microorganism reporting lists can also be configured.

Read QC
• If checked, reads are pre-processed using quality metrics before analysis.
• If unchecked, read quality metrics are calculated, but no reads are changed before analysis.

Bacterial AMR Markers reported only when an associated microorganism is detected

Bacterial AMR (antimicrobial resistance) marker reporting with associated microorganisms

• If checked, detected bacterial AMR markers are reported if the bacterial AMR marker passes a minimum detection threshold and one or more associated microorganisms are also detected and reported.
• If unchecked, detected bacterial AMR markers are reported if the bacterial AMR marker passes a minimum detection threshold.
• This option is disabled if VSP (2nd gen) or Custom Panel is selected.

AMR Only

Report AMR markers only Reporting Bacterial AMR markers only.

• If checked, only AMR markers and no microorganisms reported.
• This option is disabled if VSP (2nd gen) or Custom Panel is selected.
• This option is disabled if the Bacterial AMR marker reporting with associated microorganisms option is enabled.

Report microorganisms and/or AMR that are below threshold

Below Threshold Reporting

• If checked, microorganisms and/or AMR markers below detection thresholds are included in the reports.
• If unchecked, only microorganisms and/or AMR markers above detection thresholds are included in the reports.
• This option is disabled if Custom Panel is selected.
• This option is disabled if the Bacterial AMR marker reporting with associated microorganisms option is enabled.

Nextclade

Run Nextclade on applicable consensus genomes. This option is not applicable for UPIP and Custom test types.

Quantitative Internal Control (IC)

Select a quantitative Internal Control (IC) from the available list. If the quantitative IC is set to NONE, the IC concentration value is ignored. An incorrect quantitative IC or incorrect IC concentration results in inaccurate microorganism absolute quantification results.

Internal Control Concentration

Select a quantitative Internal Control (IC) from the available list. The number must be normalized (ie, one digit left of the decimal).

Examples: 1.2x10⁷, 3.14159x10³

If the quantitative IC is set to NONE, the IC concentration value is ignored.

Sample ID

Used for setting Per Sample Configuration to configure how individual samples are analyzed.

Control Type

Set to configure how individual samples are analyzed.
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:
• 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.