Denature and Dilute Libraries for the MiSeq system

This procedure denatures and dilutes libraries to a final volume of 600 µl. The recommended loading concentration varies depending on the version of MiSeq Reagent Kit used for the sequencing run. In practice, loading concentration can vary depending on library preparation and quantification methods. Refer to the protocol for additional information.

Low diversity libraries are libraries where a significant number of the reads have the same sequence. This lack of variation shifts the base composition because the reads are no longer random.

For example, low diversity can occur with some expression studies with > 25% of one type of transcript, low-plexity amplicon pools, adapter dimer, or bisulfite sequencing. A higher concentration spike-in of PhiX helps balance the overall lack of sequence diversity.

Document	Date	Description of Change
Document # 15039740 v11	January 2024	Converted to HTML format. Split out protocols and other information to separate documents and created a dynamic protocol generator with links to those documents.
Document # 15039740 v10	February 2019	Replaced Suggested Final Loading Concentration table in Protocol C with a single suggested concentration range.
Document # 15039740 v09	November 2018	Fixed AmpliSeq for Illumina Myeloid Panel pooling ratio in Protocol D.
Document # 15039740 v08	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 # 15039740 v07	October 2018	Added Protocol D for denaturing and diluting libraries prepared using the AmpliSeq Library Equalizer for Illumina workflow.
Document # 15039740 v06	July 2018	Added pooling ratio for AmpliSeq Myeloid Panel for Illumina.
Document # 15039740 v05	May 2018	Removed caution against using PhiX with Protocol C.
Document # 15039740 v04	April 2018	Added Protocol C for denaturing and diluting AmpliSeq for Illumina Panels.
Document # 15039740 v03	December 2017	Added recommendation in Protocol A to reference the Nextera DNA Flex Library Prep Reference Guide when working with the Nextera DNA Flex Library Prep Kit.
Document # 15039740 v02	February 2017	Added loading concentration recommendations for TruSeq Myeloid Sequencing Panel, TruSeq Custom Amplicon v1.5, and TruSeq Custom Amplicon Low Input Sequencing Kit.
Document # 15039740 v01	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.
Part # 15039740 Rev. D	November 2013	Added recommendation for low diversity libraries to dilute PhiX control libraries to the same concentration as denatured sample libraries.
Part # 15039740 Rev. C	August 2013	Added recommendation to use molecular biology grade NaOH. Added recommended library denaturation and PhiX control protocols for use with MiSeq Reagent Kit v3. Removed loading samples library information. That information is now in the MiSeq System User Guide (part # 15027617).
Part # 15039740 Rev. B	March 2013	Reduced PhiX recommendations for low diversity libraries from ≥ 25% to ≥ 5%. This change is possible when using RTA 1.17.28, or later, released with MCS v2.2. Corrected the resulting NaOH concentration for denatured 10 pM library to 1 mM. Updated instructions for combining prepared libraries and PhiX control to total 600 µl.
Part # 15039740 Rev. A	January 2013	Initial release.