Index color balancing for the NextSeq 1000/2000 system using standard SBS reagents

When pooling libraries for sequencing on the NextSeq 1000/2000 system using standard SBS reagents, it is important to select an appropriate index combination to avoid cluster registration failures during the index read(s). This articles provides guidelines for pooling Illumina libraries on the NextSeq 1000/2000 system using standard SBS reagents. For guidance using XLEAP SBS reagents, see Index color balancing for XLEAP SBS reagents on the NextSeq 1000/2000 and NovaSeq X/X Plus.

NextSeq 1000/2000 2-Channel Sequencing

The NextSeq 1000/2000 system utilizes two dyes and two images to encode data for the four nucleotides. The NextSeq 1000/ 2000 uses one image from the green channel and one image from the blue channel, while other two-channel systems (such as the NextSeq 500/550) use one image from the green channel and one image from the red channel.

NextSeq 1000/2000 2-channel sequencing with standard SBS kits uses two fluorescent dyes in four combinations: Blue for C, Green for T, Green and Blue for A, and no dye for G.

Figure 1: NextSeq 1000/2000 2-channel standard SBS fluorescent imaging. Clusters with intensity in green are T-bases. Clusters with intensity in blue are C bases. Clusters with intensity in green and blue are A bases. Clusters with no intensity/dark are G bases.

Index Considerations

• Index reads must begin with at least one base other than G in either of the first two cycles. Signal must be present in either of the first two index cycles to ensure successful sequencing. If an index read begins with two G bases, no signal intensity is generated, and cluster registration will fail.

• Select index sequences that provide signal in at least one channel, preferably both channels, for every cycle.

  • Blue channel - A or C

  • Green channel - A or T

Index Combinations Examples

Ideal index combinations: contain signal in both channels for every cycle.

Acceptable index combinations: have signal in only one channel, but there is still enough signal to sequence.

Unacceptable index combinations: will fail registration because the index read begins with 2 base calls of G and no signal intensity is generated.

Additional index combinations can be reviewed here.

For any feedback or questions regarding this article (Illumina Knowledge Article #3339), contact Illumina Technical Support [e-mail].