Sequencing Human Whole Genomes on the NovaSeq 6000 System

Workflow Overview

This page provides information and recommendations for performing Human Whole-Genome Sequencing (WGS) on the NovaSeq 6000 Sequencing System.

Library Preparation

The TruSeq DNA PCR-Free library prep protocol provides the following capabilities for whole-genome sequencing.

• Low sample (LS) and high sample (HS) protocols accommodate a range of experiments.
• A PCR-Free workflow that increases genome coverage quality to detect long repeat expansions.
• IDT for Illumina-TruSeq Unique Dual (UD) Indexes are available for purchase separately.

For information on the TruSeq DNA PCR-Free procedures and additional features, see the TruSeq DNA PCR-Free Reference Guide.

DNA Input Requirements

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.

Low and High Sample Size

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. 

IDT for Illumina – TruSeq UD Indexes

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. 

Library Quantification

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.

Sequencing and Analysis

The NovaSeq 6000 System provides the following capabilities for whole-genome sequencing.

• The NovaSeq 6000 is a dual flow cell system with a broad output range.
• The NovaSeq Xp flow cell dock allows sequencing of different libraries in each flow cell and decreases loading volume.
• Patterned flow cells reduce spacing between nanowells to increase cluster density and data output.
• Real-Time Analysis (RTA) performs image analysis and base calling during a run.
• The sequencing workflow integrates with BaseSpace Sequence Hub.
• Separate projects or methods by lane.
• Multiplex within a lane to maximize the total number of samples per flow cell.
• Complete the NovaSeq 6000 System protocol with less library input.

Requirements

View the NovaSeq 6000 Sequencing System Site Prep Guide for operation and install requirements.

Adjustable Output

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.

NovaSeq Xp Workflow

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:

• Separate projects or methods by lane.
• Multiplex within a lane to maximize the total number of samples per flow cell.
• Complete the NovaSeq 6000 System protocol with less library input.

For full protocol information, see the NovaSeq 6000 Sequencing System Guide. For a condensed version of the NovaSeq Xp protocol, see the NovaSeq Xp Workflow Checklist.

Patterned Flow Cell Technology

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.

For more information, see the Patterned Flow Cell Technology web page, Patterned Flow Cell Technology video, and Patterned Flow Cell Technology technical note.

Real-Time Analysis

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.

Sequencing Analysis Viewer

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.

BaseSpace Sequence Hub

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.

Data Storage

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.

Additional Resources for Library Preparation

Additional Resources for Sequencing and Analysis