When pooling libraries for sequencing on the NextSeq 500/550 and MiniSeq systems, it is important to select compatible index combinations, otherwise, the Index Read sequencing can fail due to cluster registration failures. This bulletin provides guidelines for pooling Illumina libraries on the NextSeq 500/550 and MiniSeq systems.

2-Channel Sequencing

NextSeq 500/550 and MiniSeq systems use two images to determine all four base calls, one red channel and one green channel. Rather than using a separate fluorescent dye for each base, 2-channel sequencing uses combinations of two fluorescent dyes: red for C, green for T, green and red for A, and no dye for G.

Figure 1: Two-channel SBS fluorescent imaging (false color image of the dyes is shown for illustration purposes). Accelerated detection of all four DNA bases is performed on the MiniSeq and NextSeq 500/550 series systems using two images to capture red and green wavelength bands. Clusters seen only in red or green images are identified as C and T bases, respectively. Clusters observed in both red and green images are identified as A bases, while unlabeled clusters are identified as G bases.

Index Considerations

• Index Reads must begin with at least one base other than G in either of the first two cycles. If an Index Read begins with two base calls of G, no signal intensity is generated, and cluster registration will fail. Signal must be present in either of the first 2 cycles to ensure demultiplexing performance.
• Select index sequences that provide signal in at least one channel, preferably both channels, for every cycle.
  • Red channel - A or C
  • Green channel - A or T

This base calling process ensures accuracy for data analysis.

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 red channels.
✔= signal in only one channel, acceptable combination.
✖= signal missing in at both channels

More information about 2-channel sequencing can be found on the 2-Channel SBS Technology web page.