Understanding the differences between Illumina index chemistries and anchor function

Illumina historically offered two primary types of index adapters:

• Complete adapters that are added via ligation to A-tailed library fragments (TruSeq, Figure 1.A).

• Partial adapters that are added via PCR to library fragments that include partial adapter sequences inserted during tagmentation (Illumina DNA/RNA UD Indexes, Tagmentation and IDT for Illumina DNA/RNA UD Indexes, Tagmentation, Figure 1.B).

Note: IDT for Illumina DNA/RNA UD Indexes were formerly named Nextera UD indexes, and the DNA/RNA UD, Tagmentation 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 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, DNA/RNA UD Indexes are added in a PCR step (Figure 1.C.ii), as also seen with the Tagmentation workflow (Figure 1.B.ii). The benefit of this approach is that it enables libraries with ligation chemistry to use the 384 Illumina DNA/RNA UD Indexes or 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 RNA UD Index, Ligation kits include one DNA/RNA UD Index plate and one RNA Index Anchor plate. In contrast, DNA/RNA UD Index, Tagmentation kits only include the index plate. For a complete list of catalog numbers and their components, see the article Catalog/Reference numbers for IDT for Illumina DNA/RNA UD and Illumina DNA/RNA UD Indexes.

For the 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 UD Indexes are not the same as the IDT for Illumina UMI DNA/RNA UD Indexes Sets A or B, Ligation (catalog numbers: 20034701 (A), 20034702 (B), 20066404 (A for automation), and 20063213 (B for automation)), or the UMI Index Anchors 3 (UMI3) that are part of the TSO500 v2 library prep kit (reference numbers: 20126462 (regular), and 20126380 (automation)). For more details, see Overview and comparison of Illumina Unique Molecular Identifier (UMI) and anchor kit options.

For any feedback or questions regarding this article (Illumina Knowledge Article #6561), contact Illumina Technical Support [email protected].