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


Along with other best practices, using the unique dual indexing strategy will make sure that your libraries will 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.

What you need to know about Unique Dual Indexing:

  • Unique dual indexing is a sequencing strategy that has distinct, unrelated index sequences for the i5 and i7 Index Read.
  • 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 the white paper Effects of Index Misassignment on Multiplexing and Downstream Analysis.
  • Unique dual indexes, or Non-Redundant Indexes, are used in the ligation step without any changes to the library preparation workflow.
  • In contrast to combinatorial dual indexing, unique dual indexing has completely unique indexes (96 unique i5s and 96 unique i7s). With combinatorial dual indexing, there is a limit to 8 unique dual pairs, resulting in the majority of the libraries sharing common indexes on the i5 or i7 end.


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



  • TruSeq DNA encompasses TruSeq DNA PCR-Free, TruSeq DNA Nano, and TruSeq DNA Exome.
  • TruSeq RNA encompasses TruSeq Stranded mRNA and TruSeq Stranded Total RNA. Nextera compatible UDI’s are expected late 2018.

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