What index combinations should I select when sequencing low plex pools of 10x Genomics Libraries generated with the Dual Index TT or Dual Index TS plates?

Question: What index combinations should I select when sequencing low plex pools of 10x Genomics Libraries generated with the Dual Index TT or Dual Index TS plates?

Answer: This article contains suggested index combinations to maintain nucleotide diversity when sequencing low plex pools of libraries (2 to 4-plex pools) prepared using Dual Index Plate TT or TS. 

For optimal run performance and optimal demultiplexing results, Illumina recommends ensuring there is sufficient nucleotide diversity in the index reads. For sequencing platforms with two channel chemistry, Illumina recommends selecting index sequence combinations such that signal will be present in both channels for each index cycle. 

Please refer to the following Illumina resources:

• What is nucleotide diversity and why is it important?
• Index color balancing for XLEAP SBS reagents on the NextSeq 1000/2000 and NovaSeq X/X Plus.
• Index color balancing for the NovaSeq 6000 system
• Index color balancing for the NextSeq 1000/2000 system using standard SBS reagents

Presented in the tables below are suggested index combinations when sequencing low-plex pools on 2-channel systems. 

The following criteria were used for selecting these index combinations:

• For 2-channel systems with XLEAP-SBS chemistry (e.g. NovaSeq X Series, NextSeq 1000/2000 XLEAP-SBS), each cycle of the index read should contain at least one C or T. This follows Illumina's guidance to avoid index combinations that have signal in the blue channel only (from A or A+G) or no signal (only G)
• For 2-channel systems with non-XLEAP SBS chemistry (e.g. NovaSeq 6000, NextSeq 2000 standard SBS), each cycle of the index read should contain at least one non-G base. This follows Illumina's guidance of selecting index combinations that provide signal in at least one channel, preferably both channels (blue channel - A or C; green channel - A or T)

These index combinations are suggested theoretical combinations to optimize color balance. 

• Not all index combinations have been specifically tested. 10x Genomics has not performed extensive testing of low plex pools.
• The tables below are not exhaustive and may not cover all possible index combinations. 

The guidance below is for 10x Genomics libraries prepared using Dual Index Plates TT or TS. Refer to this linked article for a list of library types that are prepared using Dual Index Plates TT or TS. Currently, we have not generated a list of suggested low plex index combinations for 10x Genomics Dual Index Plates NT, NN, and TN. These Index Plates are used for Feature Barcode libraries (e.g. Cell Surface Protein, CRISPR, etc.), which we do not recommend sequencing alone. Feature Barcode libraries should be pooled with their associated Gene Expression libraries for sequencing.

The guidance below is for instruments with 2-channel chemistry. Currently, we have not generated a list of suggested index combinations for other Illumina instruments. Please refer to the above Illumina resources for general guidance on color balancing recommendations for different Illumina instruments. 

Additional note on 10x Genomics index plate design: 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 Genomics Dual Index Plates were designed such that none of the i5 or i7 index sequences begins with “GG”.

For optimal demultiplexing results, accurate library quantification and appropriate loading concentrations are also critical, as discussed in this article: How do alternative quantification methods other than qPCR impact library loading?

Table 1: Recommended 2-plex, 3-plex, and 4-plex pooling for 2-channel systems with XLEAP-SBS chemistry (e.g. NovaSeq X Series, NextSeq 2000 XLEAP-SBS)

Table 2: Recommended 2-plex, 3-plex, and 4-plex pooling for 2-channel systems with non-XLEAP SBS chemistry (e.g. NovaSeq 6000, NextSeq 2000 standard SBS)

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