Chemistry and Imaging on NovaSeq 6000

The NovaSeq 6000 uses two-channel chemistry and Patterned Flow Cells technology.

Two-Channel SBS Chemistry, rather than using a separate dye for each base like the Four-Channel SBS Chemistry, two-channel SBS simplifies nucleotide detection by using two fluorescent dyes and two images to determine all four base calls (Figure1).

Figure1: Two-Channel SBS Chemistry by schematic representation.

Images are taken using red and green filter bands. Thymines are labeled with a green fluorophore, cytosines are labeled with a red fluorophore, and adenines are labeled with both red and green fluorophores. Guanines are permanently dark (Figure 2). Nucleotides are identified by analysis of the different emission patterns for each base across the combination of Image 1 and Image 2 that are processed by image analysis software to identify which bases are incorporated at each well position.

Figure 2: Image analysis during SBS Chemistry with Two-Channel Detection.

The system uses a patterned flow cell with nanowells (Figure 3) with 4 different flow cell options which allow the NovaSeq output to scale to different applications and throughput levels.

Clustering and sequencing occur in the nanowells with a proprietary ExAmp chemistry to ensure that each well in the flow cell generates a single clonal cluster.

Figure 3: Schematic representation of NovaSeq 6000 flow cell and the nanowells.

The NovaSeq Flow Cell specifications are further detailed in the table below.

For more information, see the Sequencing by Synthesis page here and the NovaSeq 6000 Sequencing System Guide here.

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