Illumina sequencers using the patterned flowcell technology (NovaSeq X, iSeq, NextSeq 2000, HiSeq 4000 &X Ten) can show an increased rate of barcode switching events. These artifacts are enabled by the exclusion amplification chemistry used on these sequencers; IIlumina calls the artifacts “index hopping”. Please see the index hopping information from Illumina here and in this video from Illumina. We have also have posted additional information here.
Please note that the AVITI sequencers, the MiSeq sequencer and NextSeq 500 chemistries are not sensitive to index hopping. For these sequencers single-indices and combinatorial indexing are fine.
To best avoid barcode switching read mis-assignments on new Illumina sequencers two measures are recommended:
- The use of uniquely dual-indexed (UDI) adapters. Illumina has collected information on the design of such adapters here. This adapter design employs unique barcodes for both index 1 and index 2 for each of the pooled libraries. Thus, 96 samples will use 96 different index 1 adapter sequences and 96 different index 2 adapter sequences.
- The efficient removal of any free primers and adapter-dimers from the libraries
The use of UDI adapters is highly recommended especially for the NovaSeq. Hereby, matching i5 and i7 indices per library must be avoided. UDI adapters can also be used on the MiSeq and the NextSeq, but they do not offer any significant advantages on these sequencers that employ the bridge-amplification chemistry. Further, any traces of free primers, primer-dimers, and adapter dimers should be removed from the sequencing libraries or the pools.
Commercial sources of UDI adapters: TruSeq-style uniquely indexed adapters are available from both Illumina and BiooScientific. Qiagen, NEB, and NuGEN are also supporting their library prep kits with optional UDI adapters. Nextera style indices need to be custom ordered from oligo vendors.
The DNA Technologies Core has 96-plex UDI adapter sets in stock that can be added to sequencing libraries by PCR. These barcode sets are available for both Nextera and TruSeq adapter designs. Please note that for TruSeq style libraries one will need to ligate a shortened and index-less stub-adapter instead of a standard Illumina adapter. The indices are then added after the cleanup of the ligation reaction by PCR.
Sequencing libraries prepared by the DNA Technologies Core:
The DNA Technologies Core uses UDI adapters for all library prep protocols that are compatible with dual indexing (e.g. DNA-Seq, RNA-seq, 3′-Tag-Seg, WGBS-Seq, …). The Core also makes sure to remove any traces of free primers, primer-dimers, and adapter-dimers.
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