This page provides information and recommendations for performing Human Whole-Genome Sequencing (WGS) on the NovaSeq 6000 Sequencing System.
The TruSeq DNA PCR-Free library prep protocol provides the following capabilities for whole-genome sequencing.
For information on the TruSeq DNA PCR-Free procedures and additional features, see the TruSeq DNA PCR-Free Reference Guide.
The TruSeq DNA PCR-Free kit requires high-quality gDNA. Degraded DNA can result in low yields and sample loss during bead cleanup. For more information on DNA input recommendations, quantifying input DNA, and assessing DNA quality, see the TruSeq DNA PCR-Free Reference Guide.
The TruSeq DNA PCR-Free contains protocols for both low sample (LS) and high sample (HS) sizes. The LS protocol is optimized for processing 24 samples at one time and the HS protocol is optimized for processing 96 samples at one time.
For information on each protocol and consumable information, see TruSeq DNA PCR-Free Reference Guide.
The IDT for Illumina-TruSeq UD Indexes increase plexity to allow accurate read assignment and efficient flow cell usage. IDT for Illumina-TruSeq Indexes are purchased separately. For information on pooling guidelines, see the Index Adapter Pooling Guide. For information on the index adapter sequences, see Illumina Adapter Sequences.
Achieving high-quality data on Illumina sequencing systems requires optimum cluster density across every lane of the flow cell. Optimizing cluster densities requires accurate quantification of PCR-free DNA libraries using qPCR. Quantification by methods other than qPCR might be inaccurate. Non-qPCR methods quantify molecules that do not have adapters on both ends and do not form clusters. More of these nonclustering molecules can be present due to the absence of PCR enrichment.
For more information on quantifying PCR-free DNA libraries, see the TruSeq DNA PCR-Free Reference Guide.
The NovaSeq 6000 System provides the following capabilities for whole-genome sequencing.
View the NovaSeq 6000 Sequencing System Site Prep Guide for operation and install requirements.
The NovaSeq 6000 System can sequence one flow cell, or two flow cells with different outputs simultaneously. For whole-genome sequencing using a PCR-free library prep, use a 2 x 150 bp read length with an insert size of 350 bp or a 2 x 250 bp read length with an insert size of 550 bp. For more information on sample throughput and run time for whole-genome sequencing, see NovaSeq System Specifications.
The NovaSeq Xp workflow is an alternative to standard onboard cluster generation on the NovaSeq 6000 System. With the NovaSeq Xp workflow, you can perform the following additional procedures:
The NovaSeq 6000 System uses patterned flow cells to generate tightly spaced clusters. Patterned flow cells contain billions of nanowells at fixed locations across both surfaces of the flow cell. The structured organization provides even sequencing cluster spacing to make flow cells less susceptible to overloading and more tolerant to a broader range of cluster densities. Precise nanowell positioning eliminates the need to map cluster locations (template generation) and saves hours on each sequencing run. Higher cluster density outputs more usable data per flow cell to drive down the sequencing run cost.
The NovaSeq 6000 System uses RTA3 software to provide real-time metrics of run quality stored as InterOp files. InterOp files are a binary output containing tile, cycle, and read-level metrics. For more information on using RTA3, see the NovaSeq 6000 Sequencing System Guide.
The Sequencing Analysis Viewer (SAV) allows you to view real-time metrics from the InterOp files provided by RTA3. SAV can be installed on a network computer to monitor runs from a remote location. To download the latest version, access documentation, and view compatible products, see Sequencing Analysis Viewer.
The Whole Genome Sequencing app on Basespace Sequence Hub is a tool for WGS data that supports alignment, variant calling, and annotation. For more information on the Whole-Genome Sequencing app, see the BaseSpace Sequence Hub Whole Genome Sequencing App.
After analysis, you can use BaseSpace Variant Interpreter to identify biologically significant variants with readily accessible annotations. If rapid analysis of small variants is a priority, the DRAGEN Germline Pipeline app analyzes human whole genomes at 30x coverage in about 60 minutes. For information on pipeline tools and outputs, see DRAGEN BIO-IT Platform.
Illumina recommends uploading data to BaseSpace Sequence Hub. For information on the required storage per run, see the NovaSeq 6000 Sequencing System Site Prep Guide. For information on configuring BaseSpace Sequence Hub, see the NovaSeq 600 Sequencing System Guide.
|TruSeq DNA PCR-Free Reference Guide
(document # 1000000039279)
|Provides comprehensive information on the TruSeq DNA PCR-Free kit, including a detailed protocol.|
|TruSeq DNA PCR-Free Checklist
(document # 1000000040870)
|Provides a condensed version of the TruSeq DNA PCR-Free protocol for experienced users.|
|TruSeq DNA PCR-Free Consumables & Equipment
(document # 1000000040873)
|Provides an interactive checklist of equipment and consumables used with the TruSeq DNA PCR-Free Kit.|
|Index Adapters Pooling Guides
(document # 1000000041074)
|Provides guidelines for preparing libraries with balanced index combinations for sequencing on Illumina systems.|
|TruSeq DNA PCR-Free support page||Provides documentation, training resources, frequently asked questions, and information on compatible Illumina products.|
|NovaSeq 6000 System Guide
(document # 1000000019358)
|Provides a system overview and instructions for operating and maintaining the NovaSeq 6000 Sequencing System.|
|NovaSeq Xp Workflow Checklist
(document # 1000000044404)
|Provides a condensed version of the NovaSeq Xp protocol for experienced users.|
|NovaSeq 6000 System support page||Provides documentation, training resources, frequently asked questions, and information on compatible Illumina products.|
|BaseSpace Sequence Hub Whole Genome Sequencing App||Provides online help for using the Whole Genome Sequencing app on BaseSpace Sequence Hub.|