Multiplexing UDI Primers & Adaptors (Illumina Platform)

The Multiplexing UDI Primers & Adaptors (illumina platform) is developed for multiplexing 384-1536 NGS libraries. The kit contains two types of indexes: unique dual index (UDI; i5 index and i7 index) and inline index. Multiplexing of NGS samples will reduce sequencing costs by pooling multiple NGS libraries into a single flow cell lane.

We have developed a Four-Base Difference UDI. The UDI allows us to make indexes that have at least 4 bases different from each other in the 8-base index length. Our unique dual indexing primers remove sequencing errors such as index hopping, index cross-contamination, mis-assignment of reads, amplification errors, and de-multiplexing errors. The primer set includes 96 pre-mixed unique pairs of i5 and i7 index primers in a 96-well plate (index information can be downloaded Here).

Even distribution of mulpexed samples using dual index primers.

Identification of index hopping by unique dual index.  The numbers of reads from all libraries were demultiplexed and analyzed.

• • • Demultiplexed: correct i5 and i7 index combination
    • Identified index hopping: correct barcodes, but NOT correct i5 and i7 index combination
    • Other: not fitting into the above categories

The integration of inline index allows more samples to be multiplexed. The size of the inline index is 7-8 nt. The combination of unique dual index with inline index makes it possible to multiplex 384 to 1536 samples.

Sequence of the final library with index locations:

Features

• Combination of UDI with inline index
• Multiplexing from 384 samples to 1536 samples
• Improves sample-identification specificity with 4-Base Difference UDI
  • Each index has at least 4 bases different from other indexes
  • 4-base difference greatly increases specificity
• Minimizes sequencing errors such as:
  • Index hopping
  • Index cross-contamination
  • Mis-assignment of reads
  • Amplification errors
  • De-multiplexing errors

Protocol & MSDS Download

• Protocol download
• MSDS download

Kit Components

UDI, Cat.# 30078

96 pre-mixed unique pairs of i5 and i7 index primers Inline adaptors

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