How to set OverrideCycles values for variable index sequence lengths on the NovaSeq X/X Plus

BCL Convert 4.1+ allows a per-sample OverrideCycles value for each sample in the sample sheet [BCLConvert_Data] section. Below is an example of some per sample settings that can be used in this section:

[BCLConvert_Data] Lane,Sample_ID,Index,Index2,OverrideCycles,AdapterRead1,AdapterRead2

NovaSeq X/X Plus flow cells contain 8 independent lanes. Illumina supports running different library preparation types in different lanes.

BaseSpace Sequence Hub (BSSH) Run Planning or the on-board built in run planning tool on the NovaSeq X/X Plus is recommended for setting up sample sheets for NovaSeq X/X Plus runs. Run Planning allows adding multiple configurations for runs and will automatically populate appropriate OverrideCycles values for Illumina library index adapter kits and user defined custom index adapter kits. When using multiple configurations, including a configuration with an index adapter kit with shorter index sequence lengths than the number of cycles specified for the read, an OverrideCycles value is automatically populated in the [BCLConvert_Data] section of the sample sheet and a base mask is applied to the cycles that are not part of the index sequence of the shorter index samples by default.

Run Planning allows users to select "Not Specified" for third party index adapter kits (and the index adapter kit does not need to be defined beforehand with this approach). When selecting "Not Specified" as the index adapter kit, Run Planning auto-populates an OverrideCycles value to use all cycles of the indexing reads by default for these samples; if the "Not Specified" sample index adapter sequences are shorter than the cycles used for the indexing reads, then the OverrideCycles value for these sample should be edited using rules below. It is possible for users to set up sample sheets manually without the use of Run Planning tools, if needed.

When running a sample with a shorter index2 sequence length than the specified cycles for index2, the cycles at the beginning of index2 must be masked in the OverrideCycles index2 value. Below is an example OverrideCycles value for a sample with dual 8 nucleotide index lengths for a run setup with 10 cycles for each indexing read (and 151 cycles for the sequencing reads).

OverrideCycles value: Y151;I8N2;N2I8;Y151

The NovaSeq X/X Plus v2 sample sheet settings guide highlights such an OverrideCycles example.

In general, (for dual indexed libraries) when running different index length libraries on NovaSeq X/X Plus platform, below are the recommendations for setting OverrideCycles values for masking bases in shorter length index sequence libraries.

IANB;NCID

I: Specifies an indexing read

A: length of index1 sequence

N: Specifies cycles to be masked

B: (number of cycles specified for index1 read) - (length of index1 sequence)

C: (number of cycles specified for index2 read) - (length of index2 sequence)

D: length of index2 sequence

A,B,C,and D are all placeholders here and should be set to an appropriate integer.

The RunInfo.xml files produced by NovaSeq X/X Plus runs contains IsReverseComplement="Y" for the second index read, as below.