Illumina historically offered two primary types of index adapters:

• Complete adapters that are added via ligation to A-tailed library fragments (TruSeq, Ligation, Figure 1.A).
• Partial adapters that are added via PCR to library fragments that include partial adapter sequences inserted during tagmentation (IDT for Illumina DNA/RNA Unique Dual (UD) Indexes, Tagmentation/PCR amplification, Figure 1.B).

Note: IDT for Illumina DNA/RNA UD Indexes were formerly named Nextera UD indexes, and the IDT for Illumina DNA/RNA UD Tagmentation/PCR amplification indexing strategy is also used with Nextera XT and Nextera CD indexes. AmpliSeq for Illumina indexes will not be addressed here; AmpliSeq for Illumina indexes must only be used with AmpliSeq for Illumina Library PLUS library preparation kits.

With the release of Illumina Stranded mRNA Prep, Ligation and Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus, Illumina introduced a hybrid mechanism that combines ligation and PCR amplification to add IDT for Illumina DNA/RNA UD Indexes to the libraries (Figure 1.C). In these preps, anchors containing partial adapter sequences are ligated to A-tailed cDNA (Figure 1.C.i). Then, IDT for Illumina DNA/RNA UD Index adapters are added in a PCR step (Figure 1.C.ii), as also seen with the Tagmentation/PCR amplification workflow (Figure 1.B.ii). The benefit of this approach is that it enables libraries with ligation chemistry to use the 384 IDT for Illumina DNA/RNA UD Index primers. As a result of this mechanism, Read 1 and Read 2 always begin with the base “T” (Figure 1.C.iii).

Figure 1: Three schematics depicting the differences between the three-indexing workflows.

The IDT for Illumina RNA UD Indexes Sets A and B, Ligation kits (catalog numbers 20040553 and 20040554, respectively) include one IDT for Illumina DNA/RNA UD Index plate and one RNA Index Anchor plate. In contrast, IDT for Illumina DNA/RNA UD Indexes, Tagmentation kits only include the index plate.

For the IDT for Illumina RNA UD Indexes, Ligation kits:

• The RNA Index Anchors are required for compatibility with the workflow.
• The Index Anchors are short adapters that are ligated to provide the binding sites for the index primers.
• Each well of the anchor plate is single-use and contains RNA Index Anchors.
• The anchor sequences are the same in each well of the entire plate.

Note: RNA Index Anchors are not the same as the IDT for Illumina UMI DNA/RNA UD Indexes Sets A or B, Ligation (catalog numbers 20034701 and 20034702, respectively) nor the TruSight Oncology UMI Reagents (catalog number 20024586). These UMI kit options are compatible with the TruSight Oncology 500 High-Throughput kit, or the TruSight Oncology 500 and TruSight Tumor 170 kits, respectively.