Base Calling
Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. The NextSeq 550 uses two-channel sequencing, which requires only two images to encode the data for four DNA bases, one from the red channel and one from the green channel.
Intensities extracted from an image compared to another image result in four distinct populations, each corresponding to a nucleotide. The base calling process determines to which population each cluster belongs.
Visualization of Cluster Intensities
|
Base |
Red Channel |
Green Channel |
Result |
|---|---|---|---|
|
A |
1 (on) |
1 (on) |
Clusters that show intensity in both the red and green channels. |
|
C |
1 (on) |
0 (off) |
Clusters that show intensity in the red channel only. |
|
G |
0 (off) |
0 (off) |
Clusters that show no intensity at a known cluster location. |
|
T |
0 (off) |
1 (on) |
Clusters that show intensity in the green channel only. |
