Question: Is nucleotide diversity in the index read important for Illumina sequencing?
Answer: Each well in a dual index plate (eg. Dual Index Plate TT) contains a unique i5 sequence and a unique i7 sequence.
To ensure nucleotide diversity / color balance in the i5 and i7 index reads, multiple dual index libraries with different i5 and i7 index sequences can be pooled together for sequencing.
However, when sequencing individual dual index libraries or low-plex pools of dual index libraries, there may be reduced nucleotide diversity in the index reads.
While Illumina recommends selecting library combinations that will allow for optimal nucleotide diversity in the i5 and i7 sample index reads, we have not encountered any technical issues with sequencing low-plex pools of dual index libraries, or individual dual index libraries, on NovaSeq 6000 and NextSeq 500/550 instruments.
We have not tested sequencing low plex pools or individual dual index libraries on Illumina instruments other than the NovaSeq 6000 and NextSeq 500/550. The performance of low plex pools on other Illumina instruments may vary.
* Note for 2-channel sequencing:
Illumina instruments with 2-channel sequencing require at least one base other than G for the first two cycles of each index read. To accommodate this requirement, the 10x Dual Index Plates were designed such that none of the i5 or i7 index sequences begins with “GG”.
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