When pooling libraries for sequencing on the NextSeq 1000/2000 system, it is important to select an appropriate index combination to avoid cluster registration failures during the index read(s). This bulletin provides guidelines for pooling Illumina libraries on the NextSeq 1000/2000 system.

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 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 SBS fluorescent imaging (false color image of the dyes is shown for illustrative purposes). Clusters with intensity in green are T-bases (pseudocolored black). Clusters with intensity in blue are C bases (pseudocolored green). Clusters with intensity in green and blue are A bases (pseudocolored red). Clusters with no intensity/dark are G bases (pseudocolored blue).

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 Example

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.
 

✔= signal in both channels. Note that the A call is resulting as fluorescence in both the green and blue channels.
✔= signal in only one channel, acceptable combination.
✖= signal missing in at both channels

Additional index combinations can be reviewed here.