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
- 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
- Unique dual indexes, or Non-Redundant Indexes,
are used in the ligation step without any changes to the library
- 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
- 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.