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Which sequences do I need to enter into my sample sheet for demultiplexing with Illumina software tools?

The i7 and i5 sequences for all Lexogen UDI 12 nt Unique Dual Indexing Sets (A1 - A4 and B1) are available for download at Lexogen has arranged the i5 12 nt UDI sequences for Illumina’s Forward Strand Workflow instruments (Sets A1 – A4) and Reverse Complement Workflow instruments (Set B1) for optimal use of the superior error correction functionality, see also Fig. 1 in I would prefer 8 or 10 nt UDIs instead. Is this possible?.


Lexogen’s Set B i5 sequences are physically the reverse complement of Set A. The i5 sequences of Set A and Set B are provided in the same orientation because they are given in the way the index is read out on the matching sequencing instrument. This means that UDIs of Set B used on instruments with Reverse Complement Workflow (e.g. iSeq, NextSeq or NovaSeq with v1.5 Reagent Kits) should be inserted into the sample sheet in the FWD orientation, as given in the provided file. The same applies to UDIs of Set A on instruments with Forward Strand Workflow (e.g., HiSeq, MiSeq, or NovaSeq with v1.0 Reagent Kits). Note: The use of the reverse complement sequence (“i5rc”) is only required if Set B is used on instruments with Forward Strand Workflow or vice versa for Set A on instruments with Reverse Complement Workflow.

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