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. This has been tested with 3'v3.1 Dual Index Gene Expression libraries on the NovaSeq 6000, NextSeq 500/550, and NextSeq 2000, and 3'v3.1 LT Gene Expression libraries on the MiSeq.
We have not tested sequencing low plex pools or individual dual index libraries on Illumina instruments other than the NovaSeq 6000, NextSeq 500/550, NextSeq 2000 and MiSeq. 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”.
The figure below shows examples of low plex pools sequenced on the NovaSeq 2000 instrument. Data is shown for 3'v3.1 Dual Index Gene Expression libraries that were sequenced alone (A) or sequenced in low plex pools of two libraries (B) or three libraries (C). As seen in the base composition plots from Illumina SAV software, nucleotide diversity is low for the i7 and i5 index reads (cycles 29 to 49). However, quality scores remain high throughout the run, indicating that low nucleotide diversity in the index reads does not negatively impact sequence quality.
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