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

03/19/20


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.

 

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 TruSeq, Nextera, and AmpliSeq workflows:

TruSeq Unique Dual Index kits

Product Name Catalog Number Indexes, Samples
IDT for Illumina - TruSeq DNA UD Indexes 20020590 24 Indexes,
96 Samples
IDT for Illumina - TruSeq DNA UD Indexes 20022370 96 Indexes,
96 Samples
IDT for Illumina - TruSeq RNA UD Indexes 20020591 24 Indexes,
96 Samples
IDT for Illumina - TruSeq RNA UD Indexes 20022371 96 Indexes,
96 Samples

 

Nextera Unique Dual Index kits

Product Name Catalog Number Indexes, Samples
IDT for Illumina - Nextera DNA Unique Dual Indexes, Set A 20027213 96 Indexes,
96 Samples
IDT for Illumina - Nextera DNA Unique Dual Indexes, Set B 20027214 96 Indexes,
96 Samples
IDT for Illumina - Nextera DNA Unique Dual Indexes, Set C 20027215 96 Indexes,
96 Samples
IDT for Illumina - Nextera DNA Unique Dual Indexes, Set D 20027216 96 Indexes,
96 Samples

 

Ampliseq for Illumina Unique Dual Index kits

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

 

For a complete list of Illumina adapter sequences, refer to the Illumina Adapter Sequences document.