Revision History

Clarified Nextera XT DNA Library Prep workflow overview.

Corrected normalize libraries procedure reagent name LNB1.

Clarified instructions for calculating molarity.

Clarified instructions for manual normalization.

Removed statement pertaining to incompatible TruSeq v3 primers on discontinued HiSeq 2500 System.

Updated kit options and catalog numbers.

Updated list of additional resources.

Removed references to formalin-fixed paraffin-embedded (FFPE) applications not supported by the workflow.

Removed list of acronyms.

Added HTML format.

Add IPB bead resuspension section to tips and techniques

Replaced AMPure XP with IPB Bead

Replaced Vortex verbiage with resuspend

Changed storage temperature and instructions in Clean Up Libraries preparation.

IPB bead added to consumables and acronyms sections

Added information on IDT® for Illumina®–Nextera™ UD Indexes sets A, B, C, and D, including kit contents, preparation procedures, and additional resources.

Removed plate layout information.

Removed the Pool Libraries section and moved the Check Library Quality section before the Normalize Libraries section.

Revised Additional Resources to provide more clarity on the resources available.

Revised language throughout document to provide consistency across other Nextera library preparation reference guides.

Added protocol for diluting libraries to the starting concentration.

Removed obsolesced Nextera XT Index Kit (96 Indexes, 384 Samples) (# FC-131-1002) from Kit Contents.

Added information on reviewing sequencing workflows to ensure compatibility with library prep methods.

Updated the normalize libraries procedure to indicate that shaking samples after the five-minute elution is necessary only if samples are not resuspended.

Reorganized kit contents information, including renaming some sections to match kit labeling and identify storage temperature.

Corrected the diagram that shows how the Nextera XT assay works to clarify each transposome dimer has two of the same adapter color.

Added the following information:

Added the following technical notes to the list of additional resources:

Consolidated steps in the pool libraries procedure.

Identified the NaOH consumable as molecular biology grade.

Specified the use of molecular-grade water or 10 mM Tris-HCl, pH 7.5–8.5 to dilute starting material for DNA quality assessment.

Specified proceeding immediately when tagmentation is complete so that neutralization occurs while the transposome is active.

Specified a thaw time of 20 minutes for NPM (Nextera PCR Master Mix).

Updated the normalize libraries procedure to apply to various sample numbers, not only 96.

Updated TCY plate to Hard-Shell 96-well PCR plate, skirted.

Updated magnetic stand supplier to Thermo Fisher Scientific.

Corrected the catalog numbers for Nextera kits provided in the introduction.

Corrected the illustration showing how the Nextera assay works.

Updated design of workflow diagram.

Renamed and combined some procedures as needed to improve continuity.

Simplified consumables information at the beginning of each section.

Revised step-by-step instructions to be more succinct.

Removed reference to obsolete Experienced User Cards and added reference to the Custom Protocol Selector.

Clarified AMPure XP bead recommendation for nonamplicon applications. See Clean Up Libraries.

Added information about normalizing low yield libraries. See Normalize Libraries.

Corrected index adapter labels on the assay diagram.

Corrected kit contents for Nextera XT DNA Library Preparation Index Kit v2 Set A (FC-131-2001) to include index N715.

Added info for new index kits that enable preparation of up to 384 indexed paired-end libraries.

Updated DNA Input Recommendations for diluting starting material and the potential results of incomplete tagmentation.

Added new Nextera XT Quality Metrics with new information on how to troubleshoot fluctuations in cluster density.

Removed Dual Indexing Principle and Low Plexity Pooling Guidelines sections. This information can be found in the Nextera Low-Plex Pooling Guidelines Tech Note on the Nextera XT DNA Library Prep support page.

References to read lengths on the MiSeq were updated for v3 chemistry.

Added instructions for alternate tip if processing fewer than 24 samples while transferring LNB1 beads in Library Normalization.

Added NaOH 1N pH > 12.5 to the Consumables and Equipment list as a user-supplied consumable.

Removed Tween 20 from Consumables and Equipment list. Consumable not used in protocol.

Modifications were added in PCR Clean-Up for 2x300 runs on the MiSeq.

New section for clustering samples on the HiSeq, HiScanSQ, and GAIIx. See Clustering Samples for HiSeq, HiScanSQ, and GAIIx.

The Dual Indexing Principle section listed incorrect catalog numbers for the Nextera XT Index kits. The correct catalog numbers are now listed.

Emphasized making sure the NT (Neutralize Tagment Buffer) and LNS1 (Library Normalization Storage Buffer 1) reagents are at room temperature before use in the protocol.

Removed reference to Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20 from the User-Supplied Consumables table because it is not used in this library preparation.

Emphasized making sure the NT (Neutralize Tagment Buffer) and LNS1 (Library Normalization Storage Buffer 1) reagents are at room temperature before use in the protocol.

Removed reference to Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20 from the User-Supplied Consumables table because it is not used in this library preparation.