Denature and Dilute Libraries for the NovaSeq 6000 Sequencing System
This guide explains how to denature and dilute prepared libraries for sequencing on the Illumina NovaSeq 6000 Sequencing System. It is intended to be used with the NovaSeq 6000 System Guide (document # 1000000019358). The NovaSeq 6000 system pages on the Illumina support site provide additional resources, including training, compatible products, and other considerations. Always check the support site for the latest versions.
Before You Begin
Library Guidelines
All instructions apply to supported library prep methods and assume an insert size typical for supported NovaSeq 6000 applications.
| • | For best results, pool and denature libraries for immediate sequencing. |
| • | Dilute the library to a loading concentration appropriate for the application. A loading concentration that is too low or too high negatively impacts the percentage of clusters passing filter (%PF). A low library concentration increases sequencing duplicates. An overly high library concentration depresses %PF. Refer to the protocol for additional information. |
| • | Achieving optimal %PF requires accurate library quantification and proper quality control. For recommendations, refer to the documentation for your library prep kit. |
| • | For Xp protocols, load an empty library tube into position #8 of the cluster cartridge before you set up the sequencing run. The empty library tube is used to prepare the conditioning mix before distribution to the flow cell. The conditioning mix helps boost clustering efficiency for sequencing. |
Best Practices
For best results, begin thawing the SBS and cluster cartridges before denaturing and diluting libraries. For instructions, refer to NovaSeq 6000 System Guide (document # 1000000019358).
Protocol
Introduction
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Consumables and Equipment
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PhiX
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Next Steps
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Revision History
Revision History - Protocol
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Revision History - PhiX
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Revision History - NovaSeq 6000 Sequencing System
Document
Date
Description of Change
Document # 1000000106351 v06
January 2024
Split out protocols and other information to separate documents and created a dynamic protocol generator with links to those documents.
Document # 1000000106351 v05
October 2023
Added Protocols I and J for TruSight Oncology 500 ctDNA v2 Standard and Xp loading methods.
Reorganized content for protocol variations to improve navigation.
Added HTML format.
Document # 1000000106351 v04
June 2022
Added Protocols G and H for TruSight Oncology 500 HRD Standard and Xp loading methods.
Document # 1000000106351 v03
November 2020
Added information on index cycles when sequencing TruSight Oncology 500 High Throughput Libraries.
Document # 1000000106351 v02
July 2020
Added information in support of the NovaSeq 6000 Reagent Kit v1.5.
Document # 1000000106351 v01
March 2020
Added Protocols E and F for TruSight Oncology 500 HT Standard and Xp loading methods.
Document # 1000000106351 v00
January 2020
Initial release.