When pooling libraries for sequencing on the NovaSeq system, select the correct index combination to avoid cluster registration failures of the Index Read. This bulletin provides guidelines for pooling Illumina libraries on the NovaSeq system.

2-Channel Sequencing

The NovaSeq system requires two images to determine all four base calls: one red channel and one green channel image. Rather than using a separate fluorescent dye for each base, 2-channel sequencing uses two fluorescent dyes in four combinations: Red for C, Green for T, Green and Red for A, and no dye for G.

Figure 1: 2-channel SBS fluorescent imaging (false color image of the dyes is shown for illustrative purposes).  Detection of all four DNA bases is performed on the NovaSeq system using only two images to capture red and green wavelength bands. Clusters seen only in red or green images are interpreted as C and T bases, respectively. Clusters observed in both red and green images are flagged 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 two cycles to ensure successful demultiplexing.
• 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 provide sufficient signal for successful sequencing.
 

Unacceptable index combinations will fail registration due to a lack of signal resulting from the Index Read begins with two G bases.
 

✔= 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.