Revision History

Document #

Date

Description of Change

1000000041074 v17

July 2026

• Corrected two-plex through eight-plex pooling strategies for NovaSeq X Series or NextSeq 1000/2000 with XLEAP-SBS chemistry on Illumina UD Indexes Set A and Set B.
• Corrected nine-plex pooling strategies for Illumina UD Index Set A and Set B, and their example figures.
• Corrected two-plex through eight-plex pooling strategies for NovaSeq X Series or NextSeq 1000/2000 with XLEAP-SBS chemistry on TruSeq UD Indexes.
• Redesigned colored layout examples.

1000000041074 v16

December 2025

• Clarified current XLEAP-SBS chemistry recommendations are only applicable to NovaSeq X Series and NextSeq 1000/2000 instruments.
• Added MiSeq i100 Series chemistry clarifications.
• Added 2 plex information for Illumina Single Cell Prep UDI Mix Strip and Unique Dual Indexes.
• Corrected index B09 of Ampliseq CD Indexes for Illumina Set C.
• Corrected index B09 of IDT for Illumina UD Indexes Set C.
• Removed references to HiSeq Systems and IEM.

1000000041074 v15

March 2025

Added Illumina Single Cell RNA Indexes.

1000000041074 v14

January 2025

• Clarified pooling strategies for instruments using XLEAP-SBS chemistry.
• Added MiSeq i100 Series information.

1000000041074 v13

February 2024

Added NextSeq 1000/2000 XLEAP-SBS chemistry information.

1000000041074 v12

September 2023

• Added NovaSeq X Series information.
• Added plate layout and pooling strategies for Illumina UD Indexes, Set A-D.
• Added clarification for V2 indexes used with IDT for Illumina UD Indexes Set C and Set D.
• Reorganized the following content to improve continuity and navigation:
• Color balance and index adapter combinations
• AmpliSeq CD Indexes
• IDT for Illumina TruSeq UD Indexes

1000000041074 v11

April 2021

• Added HTML format.
• Corrected Plexity 6 [Option 1] details in pooling strategy tables for IDT for Illumina UD Indexes and Nextera DNA CD Indexes (96 Indexes, 96 Samples).

1000000041074 v10

July

2020

• Replaced UDI0015, UDI0016, UDI0055, UDI0056, UDP0252, UDP0258, UDP0289, UDP0290, UDP0291, and UDP0301 with V2 versions.
• Added IDT for Illumina-PCR UD Indexes Set 1, 2, 3, 4.

1000000041074 v09

March 2020

• Generalized references to NextSeq Systems to include all versions.
• Added NextSeq 2000 information to Two-Channel Chemistry section.
• Added Instrument Run Setup to Color Balance section.

1000000041074 v08

October 2019

• Corrected the third option for TruSeq RNA Single Indexes Set B four-plex pooling strategy.
• Added index plate layouts for IDT for Illumina TruSeq–UD Indexes (24 Indexes, 96 Samples).

1000000041074 v07

May 2019

• Added index plate layouts for IDT for Illumina Nextera DNA UD Indexes Sets B–D.
• Identified the IDT for Illumina Nextera DNA UD index plate layout as IDT for Illumina Nextera DNA UD Indexes Sets A.
• Updated section on TruSeq Small RNA indexes to improve clarity and accuracy.

1000000041074 v06

February 2019

• Added index plate layouts for AmpliSeq CD Indexes Sets B–D for Illumina.
• Identified the AmpliSeq for Illumina index plate layout as AmpliSeq CD Indexes Set A for Illumina.

1000000041074 v05

December 2018

Corrected the Index 1 (i7) index adapter in well C2 of the AmpliSeq CD Indexes for Illumina plate to Q7008.

1000000041074 v04

November 2018

• Combined, renamed, and reorganized all sections to improve continuity, facilitate navigation, and eliminate redundancy.
• Consolidated graphics depicting pooling strategies for IDT for Illumina Nextera DNA UD Indexes and Nextera DNA CD Indexes (96 Indexes, 96 Samples).
• Added pooling guidelines for Nextera XT Index Kit v2 and Nextera Index Kit.
• Added information on Illumina Experiment Manager, Local Run Manager, and BaseSpace Sequence Hub.
• Added good and bad examples of color balance on a four-channel system.
• Identified all Index 1 (i7) and all Index 2 (i5) adapters for Nextera DNA CD Indexes (24 Indexes, 24 Samples).
• Updated plate layouts to remove duplicate index adapter names and show the Index 1 (i7) adapters first.
• Updated descriptions of one-, two-, and four-dye chemistry.

1000000041074 v04 continued

November 2018

• Described dual indexing options: combinatorial dual (CD) and unique dual (UD) indexes.
• Described pooling strategies for TruSeq CD Indexes and updated style of the graphics (the strategies have not changed).
• Referenced Indexed Sequencing Overview (document # 15057455) for indexing workflows on Illumina sequencing systems.
• Clarified the following points:
• TruSeq CD Indexes replaced TruSeq HT Indexes.
• For two-plex, dual-indexing with TruSeq CD Indexes, pool diagonally.
• Single-indexing with TruSeq CD Indexes requires dual index adapters.
• Removed the following sequences, which are available in Illumina Adapter Sequences (document # 1000000002694):
• Index adapter sequences for Nextera DNA CD Indexes (for Nextera DNA Flex).
• Index adapter and adapter trimming sequences for AmpliSeq for Illumina.
• Corrected column-based, three-plex pooling strategy for AmpliSeq for Illumina.
• Corrected names of the following items:
• IDT for Illumina kits.
• Index adapters UDP0001–UDP0009 to include four digits.

1000000041074 v03

October 2018

• Added information on IDT for Illumina Nextera DNA UD Indexes.
• Consolidated low-plexity guidelines.
• Corrected tables for Nextera DNA Flex 96 CD Indexes.
• Removed single indexing information for IDT for Illumina TruSeq DNA UD Indexes, which is compatible with dual indexing only.
• Removed incorrect guidelines for:
• Low plexity sequencing on the NovaSeq 6000 System.
• Low throughput for Nextera DNA Flex combinatorial dual indexes.

1000000041074 v02

May 2018

Added information on one-channel sequencing for the iSeq 100 System.

1000000041074 v01

January 2018

Added pooling guidelines for AmpliSeq for Illumina and Nextera DNA Flex libraries.

1000000041074 v00

October 2017

Initial release.