Illumina TruSight Whole Genome Analysis Application

Product Information

Specifications

• Product Name: TruSight Whole Genome Analysis Application
• For: In Vitro Diagnostic Use
• Version: Document # 200049931 v00, April 2024
• Analysis Workflow: Demultiplexing, FASTQ generation, read mapping, alignment to GrCh38/hg38 human reference genome, variant calling
• Variant Callers: Small Variant Caller, CNV Caller, Repeat Expansion Detection with Expansion Hunter

Product Usage Instructions

Getting Started
Ensure the TruSight Whole Genome Analysis Application is installed on the NovaSeq 6000Dx instrument. It can be found on the Applications screen of the instrument or in Illumina Run Manager on a networked computer.

Contact your local Illumina Field Representative for assistance with installation.

Data Storage Requirements
Refer to the NovaSeq 6000Dx Product Documentation and DRAGEN Server for NovaSeq 6000Dx Product Documentation for information on data output and storage.
The application outputs data into a Run Folder and an Analysis Folder in external storage. Approximate storage requirements are provided based on the size of data output for different flow cell configurations.

Approximate Analysis Time
The analysis time may vary depending on the specific parameters and workflow complexity.

FAQ

• Q: How do I access technical assistance?
  A: Please refer to the Technical Assistance section in the user manual for contact information.

• Q: What are the main functions of the TruSight Whole Genome Analysis Application?
  A: The main functions include planning sequencing runs, demultiplexing, read mapping, variant calling, quality control, andgenerating reports.

TruSight Whole Genome Analysis Application

Product Documentation

ILLUMINA PROPRIETARY Document # 200049931 v00 April 2024
FOR IN VITRO DIAGNOSTIC USE.

Revision History

This document and its contents are proprietary to Illumina, Inc. and its affiliates (“Illumina”), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s).
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Overview

The TruSight Whole Genome Analysis Application is used to plan sequencing runs for TruSight Whole Genome and automatically initiate analysis after the run completes. Analysis includes demultiplexing, FASTQ generation, read mapping, alignment to the graph-enabled GrCh38/hg38 human reference genome, and variant calling using the Illumina DRAGEN Server for NovaSeq 6000Dx.
At different stages of the analysis workflow, the application performs quality control (QC) according to defined sequencing, FASTQ, and sample library metrics, and generates reports with the results. For samples that pass all QC steps, the application generates supporting output files for use in downstream germline applications.
The TruSight Whole Genome Analysis Application executes DRAGEN variant callers, including the Small Variant Caller, Copy Number Variant (CNV) Caller, and Repeat Expansion Detection with ExpansionHunter.
The application also performs annotation of low, intermediate, or high confidence tier for small variants and includes this annotation in the output file.

Getting Started

Make sure the TruSight Whole Genome Analysis Application is installed on the NovaSeq 6000Dx instrument that will be used for sequencing as part of TruSight Whole Genome. Installed applications can be found on the Applications screen on the NovaSeq 6000Dx Instrument or in Illumina Run Manager using a browser on a networked computer. For assistance to schedule installation, contact your local Illumina Field Representative.

Data Storage Requirements
Refer to the NovaSeq 6000Dx Product Documentation (document # 200010105) and DRAGEN Server for NovaSeq 6000Dx Product Documentation (document # 200014171) for general information on data output and storage.
The TruSight Whole Genome Analysis Application outputs data into a Run Folder and an Analysis Folder in external storage. The minimum storage requirements can be approximated from the size of data output into each folder for a single sequencing run shown below.

Approximate Analysis Time
Analysis begins automatically after a sequencing run is completed and occurs sequentially on samples within a run. Data output files will be available on the external storage once analysis is complete for all samples in a run and copy transfer to the external storage is complete. When starting a sequencing run on both side A and side B at the same time, sequencing will be performed concurrently. Analysis of these sequencing runs will be performed sequentially by the TruSight Whole Genome Analysis Application after sequencing completes. The run that completes sequencing and transfer first will be analyzed first. The second sequencing run will be transferred and queued for analysis after the first analysis completes. Refer to View Run and Results on page 6 for how to determine status of active or failed runs.
Approximate time until analysis results are available after sequencing completes is shown below for the situation when side A and side B are loaded simultaneously with the same configuration.

Settings

Select the TruSight Whole Genome Analysis Application on the Applications screen to view current configuration and change permissions.

Configuration
The configuration screen displays the following application settings:

• Application Name
• Application Version
• DRAGEN Version
• RTA Version
• Release Date
• Organization
• Device Identifier
• Production Identifier
• Library Prep Kits— Displays the library prep kit. This setting cannot be changed.
• Index Adapter Kits— Displays the index adapter sets available for use.
• Index Reads
• Read Type
• Index Lengths
• Read Lengths— Read lengths are set by default when the index set is selected. This setting cannot be changed.

Permissions
The designated administrator has Permissions access and can use the checkboxes on the Permissions screen to manage user access for the TruSight Whole Genome Analysis Application.
For more information regarding permissions and user management, refer to the System Configuration section of the NovaSeq 6000Dx Product Documentation (document # 200010105).

Run Creation

Create new runs in IVD mode either on the instrument or by accessing Illumina Run Manager (IRM) using a browser on a networked computer. To access the instrument remotely, use the address and user account information provided by your Illumina representative. Refer to NovaSeq 6000Dx Product Documentation (document # 200010105) for more information.
Create Run is the recommended method for run planning. Import Sample Sheet is not recommended. The sample sheet files output in run and analysis folders are not suitable for import during run planning.

Create Runs

1. From the Runs screen, select Create Run.

2. Select the TruSight Whole Genome Analysis Application, and then select Next.

3. On the Run Settings screen, enter a run name. The run name identifies the run from sequencing through analysis.

4. [Optional] Enter a run description to further identify the run. Library Prep kit is set by default as TruSight Whole Genome and cannot be changed.

5. Select the desired TruSight Whole Genome index set from the Index Adapter Kit drop-down menu. Read length will be set by default and cannot be changed. (Read 1 and 2 use 151 cycles; Index 1 and 2 use 10 cycles).

6. Enter a Library Tube ID (recommended format as DX1234567-LIB), and then select Next.
   If no Library Tube ID is specified at this step, the planned run will need to be selected before loading of sequencing consumables. If the incorrect Library Tube ID is entered at this step, the planned run must be corrected before loading consumables. Refer to Run Revision on page 5 for protocol to correct run when ready to load consumables.

7. On the Sample Data and Sample Settings screens, sample information will be entered. Sample data can be entered manually or by importing a sample data file. The sample ID must be unique for each sample and can only contain alphanumeric characters, underscores, and dashes. Do not include spaces. Well Position refers to the well in format A01 to H04 of the index plate. Index sequence information will be populated automatically when index plate Well Position is entered. Sex must be entered as Male, Female, or Unknown. Library Plate ID and Library Well ID (eg, format A01) are required fields.

   • To enter sample data manually, add rows (to a total of 6 for S2 or 16 for S4 flow cell) and enter required information into Sample ID and Well Position Fields. Information may also be copied and pasted from Excel. Select Next. On the Sample Settings screen, enter Library Plate ID, Library Well ID, and Sex. Select Next.
   • To import a sample data file, select Import Samples and upload the sample data file. Information will be populated into rows automatically. A template (*.csv) is available for download on this screen. Select Next. On the Sample Settings screen, information will be populated into rows automatically from the imported sample data file. Select Next.

8. On the Analysis Settings screen, enter the Batch Name recorded during batch and run planning.

9. [Optional] Select the Flow Cell Type, S2 or S4.

10. Confirm or deselect the checkbox to Generate ORA compressed FASTQs, then select Next.
    NOTE The TruSight Whole Genome Analysis Application generates ORA compressed FASTQs by default. Changing this setting will increase size of final data output.

11. On the Run Review screen, review the information entered. If no changes are needed, select Save. If changes are needed, select Back as needed to return to the appropriate screen.

CAUTION
TruSight Whole Genome has been validated for 6 samples when using the NovaSeq 6000Dx S2 flow cell, and 16 samples when using the NovaSeq 6000Dx S4 flow cell. Ensure the correct number of samples are entered for the selected flow cell configuration.

Run Revision
If changes are required after run creation and before loading consumables for sequencing, revise runs in IVD mode either on the instrument or by accessing Illumina Run Manager (IRM) using a browser on a networked computer.

1. Select Runs.
2. Select the Run name on the Planned Runs tab.
3. Select Edit.
4. Update the run or sample information as needed. For example, enter or correct the Library Tube ID to match that which was used when completing the workflow.
5. Select Next up to Run Review.
6. Select Save.
7. Select Exit.

Return to Sequencing in IVD mode to repeat loading of consumables. Run should now be automatically highlighted.
If updating Library Tube ID while loading consumables, return to Run Selection in Control Software and select Refresh for the associated column, A or B. Run should now be automatically

Requeue Analysis
Refer to the Troubleshooting section in the TruSight Whole Genome Package Insert (document # 200050132) to determine which type of Requeue Analysis is most appropriate.

Requeue analysis with no changes

1. Select the Completed Run name to view Run Details.
2. Select Requeue Analysis.
3. Select Requeue Analysis with no changes.
4. Provide details in the Reanalysis Reason field.
5. Select Requeue Analysis.
6. Exit the page and navigate to the Active Runs page to confirm the requeue is in progress.

Requeue analysis with changes

1. Select the Completed Run name to view Run Details.
2. Select Requeue Analysis.
3. Select Edit run settings and Requeue Analysis.
4. Provide details in the Reanalysis Reason field.
5. Select Requeue Analysis.
6. Confirm or update the Run Settings, then select Next.
7. Correct the sample information as necessary by manually updating the fields or select Download Template to create a sampledata.csvfile with current information. Correct information and delete existing rows in the Sample Data tab before using Import Samples to populate the corrected sample data.
8. Review the information on the Run Review screen and select Save to start reanalysis.
9. Select Exit and navigate to the Active Runs page to confirm the requeue is in progress.
   The original run data folder must be present at the external storage location specified in Run Details for reanalysis to complete successfully. If reanalysis fails, make sure the run has not been moved or deleted.

View Run and Results

1. From the Illumina Run Manager main screen in IVD mode, select Runs.

2. From the Completed Runs tab, select the Run name.
   This tab will also display runs that have completed due to failure of sequencing, data transfer, or analysis. Active runs and their status are displayed in the Active Runs tab. Refer to NovaSeq 6000Dx Product Documentation (document # 200010105) for more information.

3. Select the Run name in the Completed Runs tab to view Run Details and Results for the path to the Analysis Output Folder.
   For failed runs, review the Status for each step and then refer to the Troubleshooting section of the TruSight Whole Genome Package Insert (document

   200050132).

4. Navigate to the analysis folder on your local drive and open the Consolidated Report to review the PASS/FAIL result for each QC step as follows:

   • For sequencing run QC, refer to Summary Sequencing QC Result
   • For FASTQ QC for each sample in the run, refer to Summary FASTQ QC Result
   • For library QC for each sample in the run, refer to Summary Sample Library QC Result

If a FAIL result is observed, note the QC step and refer to the Troubleshooting section of the TruSight Whole Genome Package Insert (document

200050132).

Output File Summary

The TruSight Whole Genome Analysis Application saves the following main output files. Refer to file information sections below for location of main output files.
Runs and samples which do not pass validity criteria do not produce CRAM, ROH bed, or *genome.vcf) files. QC Report Information
The Consolidated Report <>_Consolidated_Report.csvis located in the TruSightWholeGenomeAnalysis_x.x.x_run-completedirectory and contains information about quality metrics used to pass or fail samples at different stages of analysis. Individual sample reports
<>_Sample_Report.csvmay be found within the folders in the TruSightWholeGenomeAnalysis_x.x.x_run-completedirectory.

The report headers include the following information about the run: the app version, batch name, library pool tube ID, sequencing run name, sequencing run ID, and flow cell type. The following tables describe the information included in the Consolidated Report. The individual Sample Report includes the same information except for the Demultiplex Metrics.

Table 1 Sequencing QC Metrics

specification is
Q30| | set since too low %Q30 runs will not pass Q30 bases in Sample
| | Library QC.
Summary| PASS or| For Sequencing QC failure, consult Troubleshooting section in the
Sequencing| FAIL| TruSight Whole Genome Package Insert (document #
QC Result| | 200050132).

Table 2 Demultiplex Metrics

Table 3 FASTQ QC Metrics

Table 4 Sample Library QC Metrics

ensure
bases| | analytical performance.
Estimated| ≤0.005| Detects contaminating reads from other samples. Maximum
sample| | specification is set to ensure mitochondrial SNV limit of
contamination| | detection (the variant type with the highest sensitivity to
| | contamination).
Summary| PASS or| For Sample Library QC failure, consult Troubleshooting section
Sample Library| FAIL| in the TruSight Whole Genome Package Insert (document #
QC Result| | 200050132).

Table 5 Ploidy QC Metrics

Table 6 For Information Only Metrics

Variant Call File Information

VCF Files
Variant call format (*.vcf) files contain information about variants found at specific positions in a
reference genome and can be found in the /Analysis directory.
The VCF file header includes the VCF file format version and the variant caller version and lists the annotations used in the remainder of the file. The last line in the header contains the column headings for the data lines. Each of the VCF file data lines contains information about a single reference position.
All VCF files contain a header with descriptions of output columns, and variant call data in columns labeled as CHROM, POS, ID, REF, ALT, QUAL, FILTER, INFO, FORMAT, SAMPLE. Definitions of column values can vary among variant callers.
Small Variant and mSNV VCF
Output is saved under the .annotated.hard-filtered.gvcf.gzfile in the /Analysisdirectory.
A genomic VCF (gVCF) file contains information on variants and positions determined to be homozygous to the reference genome. For homozygous regions, the gVCF file includes statistics that indicate how well reads support the absence of variants or alternative alleles. The gVCF file includes an artificial allele. Reads that do not support the reference or any variants are assigned the allele. DRAGEN uses these reads to determine if the position can be called as a homozygous reference, as opposed to remaining uncalled. The resulting score represents the Phred-scaled level of confidence in a homozygous reference call. In germline mode, the score is FORMAT/GQ.
DRAGEN provides post-VCF variant filtering based on annotations present in the VCF records. Variant hard filtering is described below. However, due to the nature of DRAGEN’s algorithms, which incorporate the hypothesis of correlated errors from within the core of variant caller, the pipeline has improved capabilities in distinguishing the true variants from noise, and therefore the dependency on post-VCF filtering is substantially reduced.
The TruSight Whole Genome Analysis Application provides annotation of confidence score and confidence tier for small variants that can be used to further improve performance. Confidence tier annotation is not a quality filter and as such is not directly reflected in the quality status of the variant calls. Therefore, it is possible to see passing variant calls which are nonetheless annotated as low confidence.

Table 7 VCF File Headings

Table 8 VCF File Annotations

Copy Number Variant VCF
The target counts stage is the first processing stage for the DRAGEN CNV pipeline, producing