Understanding unique dual indexes (UDI) and associated library prep kits

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 recommends using unique dual indexing (UDI) whenever possible for the most accurate demultiplexing.

What 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

Product Name	Catalog Number	Indexes, Samples
IDT for Illumina - TruSeq DNA UD Indexes v2	20040870	96 Indexes, 96 Samples
IDT for Illumina - TruSeq RNA UD Indexes v2	20040871	96 Indexes, 96 Samples

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

Illumina DNA/RNA Unique Dual Index, Tagmentation kits

Product Name	Catalog Number	Indexes, Samples
Illumina DNA/RNA UD Indexes Set A, Tagmentation1	20091654	96 Indexes, 96 Samples
Illumina DNA/RNA UD Indexes Set B, Tagmentation2	20091656	96 Indexes, 96 Samples
Illumina DNA/RNA UD Indexes Set C, Tagmentation	20091658	96 Indexes, 96 Samples
Illumina DNA/RNA UD Indexes Set D, Tagmentation	20091660	96 Indexes, 96 Samples

• All four index sets are compatible with Nextera XT, Illumina DNA Prep3, and Illumina DNA Prep with Enrichment4, Illumina DNA Prep with Exome 2.5 Enrichment, Illumina RNA Prep with Enrichment and Illumina DNA PCR-Free.

Illumina RNA Unique Dual Indexes, Ligation kits

Product Name	Catalog Number	Indexes, Samples
Illumina RNA UD Indexes Set A, Ligation	20091655	96 Indexes, 96 Samples
Illumina RNA UD Indexes Set B, Ligation	20091657	96 Indexes, 96 Samples
Illumina RNA UD Indexes Set C, Ligation	20091659	96 Indexes, 96 Samples
Illumina RNA UD Indexes Set D, Ligation	20091661	96 Indexes, 96 Samples

• Illumina RNA UD Indexes, Ligation are compatible with Illumina Stranded Total RNA and Illumina Stranded mRNA.
• Note that these kits contain an 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 DNA/RNA Unique Dual Index, Tagmentation kits

Product Name	Catalog Number	Indexes, Samples
IDT for Illumina DNA/RNA UD Indexes Set A, Tagmentation1	20027213	96 Indexes, 96 Samples
IDT for Illumina DNA/RNA UD Indexes Set B, Tagmentation2	20027214	96 Indexes, 96 Samples
IDT for Illumina DNA/RNA UD Indexes Set C, Tagmentation	20042666	96 Indexes, 96 Samples
IDT for Illumina DNA/RNA UD Indexes Set D, Tagmentation	20042667	96 Indexes, 96 Samples

• All four index sets are compatible with Nextera XT, Illumina DNA Prep3, and Illumina DNA Prep with Enrichment4, Illumina RNA Prep with Enrichment and Illumina DNA PCR-Free.
• 1Formerly known as IDT for Illumina Nextera DNA UD Indexes Set A

• 2Formerly known as IDT for Illumina Nextera DNA UD Indexes Set B

• 3Formerly known as Nextera DNA Flex

• 4Formerly known as Nextera Flex for Enrichment

IDT for Illumina RNA Unique Dual Indexes, Ligation kits

Product Name	Catalog Number	Indexes, Samples
IDT for Illumina RNA UD Indexes Set A, Ligation	20040553	96 Indexes, 96 Samples
IDT for Illumina RNA UD Indexes Set B, Ligation	20040554	96 Indexes, 96 Samples
IDT for Illumina RNA UD Indexes Set C, Ligation	20040555	96 Indexes, 96 Samples
IDT for Illumina RNA UD Indexes Set D, Ligation	20040556	96 Indexes, 96 Samples

• 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

Product Name	Catalog Number	Indexes, Samples
IDT for Illumina UMI DNA/RNA UD Indexes Set A, Ligation	20035701	96 Indexes, 96 Samples
IDT for Illumina UMI DNA/RNA UD Indexes Set B, Ligation	20034702	96 Indexes, 96 Samples
IDT for Illumina UMI DNA/RNA Index Anchors Set A for Automation	20066404	96 Indexes, 96 Samples
IDT for Illumina UMI DNA/RNA Index Anchors Set B for Automation	20063213	96 Indexes, 96 Samples

• 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

Product Name	Catalog Number	Indexes, Samples
AmpliSeq UD Indexes for Illumina	20019104	24 Indexes, 24 Samples

• 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 knowledge article.

For any feedback or questions regarding this article (Illumina Knowledge Article #2344), contact Illumina Technical Support [e-mail].