Overview

DRAGEN Heme WGS Tumor Only Pipeline, henceforth referred as the Heme Pipeline, is a comprehensive and unbiased whole genome sequencing solution to replace conventional cytogenetic and panel sequencing approaches for detecting all types of mutation using a limited amount of DNA. It can be applied to detect clinically actionable mutations for cancer spanning a wide range of genomic events, e.g., structural variants (SV), Copy Number Alterations (CNA), small variants (SNV/insertion/deletion/delins) and internal tandem duplications (ITD) and DUX4 variants using Heme samples.

The Heme pipeline includes a DNA-only workflow designed to analyze whole genome sequencing data generated on supported instruments. It may be run as a local off-instrument solution installable on a DRAGEN server or accessible through the Illumina Connected Analytics (ICA) cloud environment. The Heme pipeline is for Research Use Only (RUO).

Features

• Superb performance based on the DRAGEN BioIT platform Release 4.4.4

• Supports starting the analysis from BCL, FASTQ, BAM or CRAM as inputs.

• Flexible custom configurable options on top of well established DRAGEN recipes for Heme WGS analysis.

• Available on local DRAGEN servers and Illumina Connected Analytics (ICA)

• Seamless integration with Illumina Connected Insights (ICI) for tertiary interpretation

Supported Library Prep Kits (LPKs)

• Illumina DNA PCR Free Prep Kit

• Illumina DNA Prep Kit

• Custom LPKs

Supported Sequencing Instruments

• NovaSeq 6000 or 6000Dx in RUO mode

• NovaSeq X or NovaSeq X plus

Note Unsupported instruments can still be analyzed, but a warning will be generated.

Supported FLow Cells

• NovaSeq 6000 or 6000Dx S4

• NovaSeq X or NovaSeq X plus 10B, 25B

The pipeline has fields that are required in addition to general sample sheet requirements. Follow the steps below to create a valid samplesheet.

Standard Sample Sheet Requirements

The following sample sheet requirements describe required and optional fields for the pipeline. Depending on the deployment (standalone DRAGEN server, ICA with auto-launch, ICA with manual launch), certain sections and required values can deviate from the standard requirements. These deviations are noted in the information below.

The analysis fails if the sample sheet requirements are not met.

Use the following steps to create a valid sample sheet.

1. Download the sample sheet v2 template that matches the instrument & assay run.

2. In the Sequencing Settings section, enter the following required parameters:

[Sequencing_Settings] Section

1. In the BCL Convert Settings section, enter the following required parameters:

[BCLConvert_Settings] Section

1. In the BCL Convert Data section, enter the following parameters for each sample.

[BCLConvert_Data] Section

1. In the [Heme_Data] section, enter the following parameters:

[Heme_Data] Section header changes depending on the deployment: Section header changes depending on the deployment:

• Standalone DRAGEN Server and ICA with Manual Launch: Heme_Data

• ICA with Auto-launch: Cloud_Heme_Data

[Heme_Data] Section

To ensure a successful analysis, follow these guidelines:

1. Avoid any blank lines at the end of the sample sheet; these can cause the analysis to fail.

2. When running local analysis using the command line save the sample sheet in the sequencing run folder with the default name SampleSheet.csv, or choose a different name and specify the path in the command-line options.

ICA with Auto-launch: Sample Sheet Requirements

Refer to the following requirements to create sample sheets for running the analysis on ICA with Auto-launch. For sample sheet requirements common between deployments see Standard Sample Sheet Requirements. Samples sheets can be created using BaseSpace Run Planning Tool or manually by downloading and editing a sample sheet template

To auto-launch analysis from the sequencer run folder, ensure the StartsFromFastq and SampleSheetRequested fields are set to FALSE. To auto-launch analysis from FASTQs after BCL Convert auto-launch, StartsFromFastq and SampleSheet Requested fields must be set to TRUE

[Cloud_Heme_Data] Section

Refer to [Heme_Data] Section for this section's requirements.

[Cloud_Heme_Settings] Section

[Cloud_Data] Section

[Cloud_Settings] Section

Description

Sample Sheet templates for the Heme pipeline for standalone DRAGEN server and ICA manual launch analysis can be found in the table below. For auto-launch compatible sample sheets, use BaseSpace Run Planner.

The Heme pipeline is compatible with several instruments and assay workflows (standard, XP), each of which have implications for the sample sheet.

Templates

Sample sheet templates contain all required fields, including index sequences in the proper orientation for all indexes from a given library prep kit. The templates are provided as a starting point for creating a sample sheet manually when launching analysis on a standalone DRAGEN server or on ICA using manual launch.

*Lane numbers cannot exceed what is supported by the flow cell in use.

Demultiplex only option

In order to break up the workflow, one may wish to run the software with the demux only option. The pipeline will perform FASTQ generation with the settings provided by default or as specified in the sample sheet. Then the subsequent analysis may start from FASTQ.

CRAM input

When CRAM is used as input, the reference genome used to generate the CRAM files is required. This may be provided using the custom configuration file

Local App Setup

Overview

This document describes how to use the Custom Configuration Support feature for the pipeline software. This feature allows users to customize a specific set of DRAGEN command-line options to override the default values pre-defined in the pipeline.

Customization with customConfig and customResourceDir

Users can customize pipeline behavior and file inputs using:

• --customConfig : path to a custom configuration file listing customized parameter values.

• --customResourceDir : path to a directory containing custom resource files.

Both options should be used together if file-based overrides are required.

Important note for using File Parameters

• For file parameters (parameters that require a file), users must specify relative paths in the customConfig file. The software will join customResourceDir and the relative path to form the full file path.

• Additionally, the value assigned to a file parameter must be enclosed in single quotes ('').

Examples

Command Line

run_Heme_WGS_TO_{version}.sh \
  --inputType bcl \
  --inputFolder /heme_input_bcl \
  --customConfig /path/heme_custom_param.config \
  --customResourceDir custom_resources_Heme_dir

heme_custom_param.config Content

# custom parameters
vc_output_evidence_bam = false
qc_detect_contamination = true
aligner_clip_pe_overhang = 0

# custom reference files
vc_systematic_noise = '/snv/WGS_hg38_v1.0_systematic_noise.snv.bed.gz'
sv_systematic_noise = '/sv/WGS_FF_Heme_hg38_v1.0_systematic_noise.sv.bedpe.gz'
vc_somatic_hotspots = '/snv/somatic_hotspots_GRCh38.vcf.gz'

custom_resources_Heme_dir Folder Structure

custom_resources_Heme/
├── snv
│   ├── WGS_hg38_v1.0_systematic_noise.snv.bed.gz
│   └── somatic_hotspots_GRCh38.vcf.gz
└── sv
    └── WGS_FF_Heme_hg38_v1.0_systematic_noise.sv.bedpe.gz

customConfig Template (with default value)

#vc_systematic_noise = ''
#enable_map_align = true
#sv_systematic_noise = ''
#vc_output_evidence_bam = false
#qc_detect_contamination = true
#vc_somatic_hotspots = ''
#sv_somatic_ins_tandup_hotspot_regions_bed = ''
#cram_reference = ''
#aligner_clip_pe_overhang = 0

Supported Parameters

ℹ️ Note: For CRAM Input Reference Genome, a list of commonly-used human reference FASTA files can be downloaded from the Illumina support site:Illumina DRAGEN Product Files