Denature and Dilute Libraries for the NextSeq 500 and NextSeq 550 Sequencing Systems

This procedure denatures and dilutes libraries to a final loading volume of 1.3 ml at a recommended concentration of 1.8 pM for high output kits and 1.5 pM for mid output kits. In practice, loading concentration can vary depending on library preparation and quantification methods. Refer to the protocol for additional information.

Document	Date	Description of Change
Document # 15048776 v19	January 2024	Split out protocols and other information to separate documents and created a dynamic protocol generator with links to those documents. Added HTML format.
Document # 15048776 v18	December 2022	Clarified list of protocol variations. Updated the protocol for denaturing and diluting TruSight Oncology 500 HRD libraries as follows: • Added list of terms and corresponding descriptions. • Clarified procedures to be used with matched sequencing runs. • Added procedures to be used with unmatched sequencing runs.
Document # 15048776 v17	June 2022	Added a new protocol for denaturing and diluting TruSight Oncology 500 HRD libraries.
Document # 15048776 v16	July 2020	Updated for internal purposes, no change to content.
Document # 15048776 v15	April 2020	Specified TruSight Oncology 500 protocols apply to all compatible TSO 500 products.
Document # 15048776 v14	March 2020	Specified NextSeq versions (NextSeq 500 and NextSeq 550) in document title.
Document # 15048776 v13	November 2019	Added the following information for TruSight Oncology 500: • Guidelines on the number of libraries and possible DNA/RNA combinations per sequencing run. • Clarification to select one of three options to pool, denature, and dilute libraries.
Document # 15048776 v12	October 2019	Added a new RNA only protocol for denaturing and diluting TruSight Oncology 500 libraries. Combined common topics in denaturation and dilution method for Protocol F. For TruSight Oncology 500, removed guidelines on the number of libraries and possible DNA/RNA combinations per sequencing run and moved the information to the TruSight Oncology 500 support pages.
Document # 15048776 v11	April 2019	Added a new protocol for denaturing and diluting TruSight Oncology 500 DNA and RNA libraries. Replaced instances of incubator with heat block in Protocols E and F. Added heat block and thermal cycler to Equipment list. Updated Denature and Dilute PhiX Control procedures to more clearly identify the compatible protocol.
Document # 15048776 v10	February 2019	Replaced Suggested Final Loading Concentration table in Protocol C with a single suggested concentration range.
Document # 15048776 v09	December 2018	Added a new protocol for denaturing and diluting TruSight Tumor 170 libraries. Added a new procedure for denaturing and diluting PhiX for TruSight Tumor 170. Added a new protocol for pooling, denaturing, and diluting TruSight Oncology 500 libraries. Added a new procedure for denaturing and diluting PhiX for TruSight Oncology 500.
Document # 15048776 v08	November 2018	Fixed AmpliSeq for Illumina Myeloid Panel pooling ratio in Protocol D.
Document # 15048776 v07	November 2018	Fixed AmpliSeq for Illumina Myeloid Panel pooling ratio in Protocol C. Added AmpliSeq for Illumina Childhood Cancer Research Assay Panel pooling ratio.
Document # 15048776 v06	October 2018	Added Protocol D for denaturing and diluting libraries prepared using the AmpliSeq Library Equalizer for Illumina workflow.
Document # 15048776 v05	July 2018	Added pooling ratio for AmpliSeq Myeloid Panel for Illumina.
Document # 15048776 v04	May 2018	Added note regarding mid output kit loading concentration. Added information on high and mid output kits. Removed caution against using PhiX with Protocol C.
Document # 15048776 v03	April 2018	Added Protocol C for denaturing and diluting AmpliSeq for Illumina Panels.
Document # 15048776 v02	January 2016	Added procedure for denaturing and diluting libraries that have been normalized using a bead-based procedure. Organized procedures as Protocol A and Protocol B. Add instructions to dilute PhiX to 1.8 pM for use as a control.
Document # 15048776 v01	October 2015	Removed extra vortex and centrifuge steps from PhiX preparation instructions. Removed instructions for using NCS v1.2 software.
Part # 15048776 Rev. E	May 2015	Changed title to the NextSeq System Denature and Dilute Libraries Guide. This guide applies to the NextSeq 500 and NextSeq 550 systems.
Part # 15048776 Rev. D	October 2014	Corrected library volume to 2995 µl when combining libraries with a PhiX spike-in and when using NCS v1.2. Added information about performing a PhiX-only run for troubleshooting purposes.
Part # 15048776 Rev. C	September 2014	Updated URL for Safety Data Sheets (SDS) to support.illumina.com/sds.html. Updated NextSeq product markings from ™ to ®.
Part # 15048776 Rev. B	August 2014	Added instructions for preparing a library loading concentration of 1.8 pM, and reduced loading volume from of 1.3 ml. This change requires (Undefined variable: Software_Name.Control_Short) v1.3. Corrected volumes for denaturing and diluting a 0.5 nM library. Updated URL for Safety Data Sheets (SDS) to support.illumina.com/sds.ilmn.
Part # 15048776 Rev. A	January 2014	Initial release.