Considerations for Index color balancing on the MiSeq i100 Series

Background

Selecting diverse index sequences that optimize color balance within pooled libraries is crucial for successful demultiplexing and data analysis. As a general guideline, Illumina recommends selecting index sequences such that signal will be present in both imaging channels for each index cycle when sequencing libraries on a 2-channel system.

Please note, even optimally color-balanced pools can display base calling errors if the run is severely overloaded or underloaded. Libraries must be sequenced at their optimal loading concentration to ensure the best demultiplexing results.

MiSeq i100 XLEAP Chemistry

While the MiSeq i100 utilizes XLEAP-SBS reagents similar to those on the NovaSeq X Series and NextSeq 1000/2000, it has some minor changes that make it unique:

• The MiSeq i100 XLEAP-SBS reagents use three fluorescent dyes and two images to encode data for the four base calls.

• The MiSeq i100 uses 'A' as the dual-color base, with 'C' as the single blue channel only base.

  

T = Green

C = Blue

A = Blue + Green

G = Dark (no label)

When sequencing with XLEAP chemistry, combine index sequences so that signal is present in both imaging channels for every cycle whenever possible. For larger pools, it is recommended to maintain base and color balance for all pooled indexes in each lane. Empirical testing is recommended to confirm index combinations not listed in Index Adapters Pooling Guide, as well as third-party or custom index combinations demultiplex reliably.

Considerations when migrating libraries from the MiSeq i100 to other XLEAP-SBS Platforms

The MiSeq i100 patterned flow cell is physically bonded to the CMOS sensor, so image registration is not required and results in superior performance when sequencing low plexity libraries. When using the MiSeq i100 to check library quality or index balance before migrating to a high-throughput system such as the NovaSeq X Series, see the following considerations.

When sequencing using XLEAP-SBS chemistry on the NextSeq 1000/2000 or NovaSeq X Series, combine index sequences so that signal is present in both channels for every cycle whenever possible. Illumina recommends avoiding index combinations which only have signal in the blue channel from A or A+G in any given cycle, or no signal (only G) present in any given cycle. It is acceptable to have signal only in the green channel from the T or C bases, if necessary.

When sequencing on a two-channel system such as the NextSeq 1000/2000 or NovaSeq X Series, either of the first two cycles of the Index Read must start with at least one base other than G. If an Index Read starts with two G bases, signal intensity is not generated and this can cause signal registration issues. For Index 1, this means that the Read cannot start with GG. The NextSeq 1000/2000 and the NovaSeq X Series sequence Index 2 in the reverse-complement orientation, thus Index 2 cannot end with CC. This criteria is more relevant for lower plexity pools as higher plexity pools are able to pass registration due to larger percentage of signal from neighboring clusters.

Considerations for Index First Sequencing

The Standard Illumina Sequencing Strategy for MiSeq i100 sequences index 2 in the reverse-complement orientation. The MiSeq i100 has the option of using Index First Sequencing. With this strategy, Indexes are sequenced before the DNA insert in the following order: Index 1 (i7), Index 2 (i5), Read 1, and lastly Read 2 after paired-end turn.

This means both Index reads are read in the forward orientation for Index First sequencing, while Index 2 is read in the reverse complement orientation in Read First sequencing. As DRAGEN Analysis software will automatically interpret the orientation based on Index First or Read First, always input the Index 2 sequences in the forward orientation. For custom or third party analysis pipelines, ensure that the Index 2 sequence is in the appropriate orientation for the pipeline.

For any feedback or questions regarding this article (Illumina Knowledge Article #9405), contact Illumina Technical Support [e-mail].