Introduction

Pre-Sequencing

Configure your instrument to send runs to BaseSpace Sequence Hub (BSSH) Run Monitoring and Storage. Prepare your sample sheet for sequencing and primary analysis (BCL Convert) using the BSSH Run Planning tool with instructions listed under Sequencing Run Setup.

Sequencing

Sequencing can be done on a NovaSeq X, NovaSeq 6000, or NextSeq 2000. For each system, the data must be converted from BCLs to FASTQs, which can be performed locally or in the cloud (BSSH or ICA). The biosamples (.fastq files) can then be used for secondary analysis. For instrument set up instructions, see the Instrument Domain Setup pages.

Secondary Analysis

Secondary analysis (alignment to a reference genome, variant calling, and more with DRAGEN) may be performed on a local DRAGEN server or in the cloud (BSSH or ICA) using DRAGEN Germline, DRAGEN Somatic, or DRAGEN Enrichment. For these instructions, view the instructions under Run Analysis Setup.

Samples may be downsampled; for more information about downsampling, please see https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-dna-pipeline/downsampling.

Tertiary Analysis

Illumina Connected Multiomics supports post-variant calling analyses, including differential methylation, combining variant regions and differentially methylated regions, and gene set enrichment analysis. For information on how to use this tool, see the instructions under Tertiary Analysis.

Additional References

For guidance on the assay workflow, please refer to the library prep documentation at the following links:

• https://support.illumina.com/sequencing/sequencing_kits/illumina-5-base-dna-prep/documentation.html

• https://support.illumina.com/sequencing/sequencing_kits/illumina-5-base-dna-prep-enrichment/documentation.html.

For data associated with the assay, including coverage recommendations, please review the 5-Base DNA Prep Data Sheet: https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/illumina-5-base-dna-prep-data-sheet-m-gl-03689/illumina-5-base-dna-prep-data-sheet-m-gl-03689.pdf

For additional information on the DRAGEN pipeline, please refer to the DRAGEN documentation at https://help.dragen.illumina.com/product-guide/dragen-v4.4/dragen-methylation-pipeline/dragen-5base-pipeline.

For additional information on the library prep kits, please review these links:

• https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/illumina-5-base-dna-prep.html

• https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/cell-free-dna-prep-enrichment.html

For additional information on genome and methylation sequencing, please review this link: https://www.illumina.com/techniques/sequencing/methylation-sequencing/methylome-genome.html