Is nucleotide diversity in the index read important for Illumina sequencing?

Question: Is nucleotide diversity in the index read important for Illumina sequencing?

Answer: Each well in a dual index plate (e.g., Dual Index Plate TT, Dual Index Plate NN, etc.) 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.

Additional information on nucleotide diversity can be found in this Illumina article: What is nucleotide diversity and why is it important.

When sequencing individual dual index libraries or low-plex pools of dual index libraries, there may be reduced nucleotide diversity in the index reads.

NovaSeq 6000, MiSeq, and NextSeq 500/550

We have tested various 10x Dual Index libraries on the NovaSeq 6000 and NextSeq 500/550, and 3'v3.1 LT Dual Index Gene Expression libraries on the MiSeq. We have not encountered any technical issues during the in-house sequencing of low-plex pools of dual index libraries or individual dual index libraries on these instruments. 

For additional guidance from Illumina, please refer to these articles:

• Best Practices for Low-Diversity Sequencing on the NextSeq 500/550 and MiniSeq Systems
• Low-Diversity Sequencing on the Illumina MiSeq Platform

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”. 

NextSeq 2000

We have received customer reports of issues demultiplexing 10x dual index libraries when sequencing on the NextSeq 2000  in both P2 and P3 flow cells with the NextSeq 2000 software NCS1.4.0 and below. The affected runs have a high % of no calls in the index read, which may lead to data loss. This issue is primarily observed in low-plexity runs and was reproduced internally.

We recommend customers take the following actions:

• Upgrade to this latest software to minimize demultiplexing issues of 10x’s dual index libraries on the NextSeq 1000 and 2000 systems. The new version of the NextSeq 1000/2000 Control Software Suite (v1.4.1) is now available for download and installation on the Illumina Support website: https://support.illumina.com/sequencing/sequencing_instruments/nextseq-1000-2000/downloads.html
• Target appropriate loading concentration. Overloaded/over clustered runs can be associated with higher % of no call. Loading concentration can be found in the Sequencing section of our User Guides.
• For older software versions, please avoid index combinations without signal in both channels for every cycle (Balanced run: A + any base OR C+T in each cycle of the index read). Additional information on color balancing on the NextSeq 2000 can be found in this Illumina Tech Note.

We have not tested sequencing low plex pools or individual dual index libraries on other Illumina instruments. The performance of low plex pools on different Illumina instruments may vary.

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