Along with best practices, using the unique dual indexing strategy will make sure that libraries sequence and demultiplex with the highest accuracy across all Illumina sequencing platforms. When preparing libraries for multiplexing, Illumina encourages customers to use unique dual indexing (UDI) whenever possible to ensure the most accurate demultiplexing.

What you need to know about Unique Dual Indexing:

• Unique dual indexing is a sequencing strategy that has distinct, unrelated index sequences for each of the i5 and i7 index reads.
• Unique dual indexing is a known mitigation for filtering index-hopped reads seen in downstream analyses. Misassigned reads will be flagged as “undetermined reads” and can be excluded from analysis. For more information about index hopping refer to Effects of Index Misassignment on Multiplexing and Downstream Analysis. Additional best practices to mitigate index hopping can be found here.
• Unique dual indexes, or non-redundant indexes, are used in the indexing step without any changes to the library preparation workflow.
• With combinatorial dual indexing, every i5 and i7 is shared among other samples on the same plate with indexes repeating down rows and across columns. The combinations are unique though the individual indexes are not.
• In contrast to combinatorial dual indexing, unique dual indexing has completely unique indexes (eg, 96 unique i5s and 96 unique i7s per 96-well plate).

Figure 1: With combinatorial dual indexing, every i5 and i7 is shared among other samples on the same plate with indexes repeating across rows and down columns; the combinations are unique though the individual indexes are not. In contrast to combinatorial dual indexing, unique dual indexing has completely unique indexes (eg, 96 unique i5s and 96 unique i7s per 96-well plate).

The following UDI kits are available for various Illumina workflows:

TruSeq Unique Dual Index kits

• TruSeq DNA UDIs are compatible with TruSeq DNA PCR-Free, TruSeq DNA Nano, and TruSeq DNA Exome
• TruSeq RNA UDIs are compatible with TruSeq Stranded mRNA and TruSeq Stranded Total RNA

IDT for Illumina DNA/RNA Unique Dual Index, Tagmentation kits

• All four index sets are compatible with Nextera XT, Illumina DNA Prep³, and Illumina DNA Prep with Enrichment⁴.
• The IDT for Illumina DNA/RNA UD Indexes Sets A-B (20027213, 20027214) are compatible with Illumina RNA Prep with Enrichment and Illumina DNA PCR-Free.

IDT for Illumina RNA Unique Dual Indexes, Ligation kits

• IDT for Illumina RNA UD Indexes, Ligation are compatible with Illumina Stranded Total RNA and Illumina Stranded mRNA.
• Note that these kits contain an IDT for Illumina DNA/RNA UD Index plate as well as an RNA Index Anchor plate. The RNA Index Anchors are required for compatibility with the workflow.

IDT for Illumina UMI DNA/RNA Unique Dual Indexes, Ligation kits

• IDT for Illumina UMI DNA/RNA UD Indexes are compatible with TruSight Oncology 500 High-Throughput.
• Note that these kits contain an IDT for Illumina DNA/RNA UD Index plate as well as an UMI DNA Index Anchor plate. The UMI DNA Index anchors are required for compatibility with the workflow.

Ampliseq for Illumina Unique Dual Index kits

• AmpliSeq UDIs are compatible with all AmpliSeq for Illumina library preparation kits

For a complete list of Illumina adapter sequences, refer to the Illumina Adapter Sequences document. For a complete list of Illumina index kits, refer to the Illumina adapter portfolio bulletin. For a comparison of unique dual indexing and combinatorial dual indexing, see our page on Dual Index Sequencing.