Phasing Correction
During the sequencing reaction, each DNA strand in a cluster extends by one base per cycle. Phasing and
prephasing occurs when a strand becomes out of phase with the current incorporation cycle.
u Phasing occurs when a base falls behind.
u Prephasing occurs when a base jumps ahead.
Figure 22 Phasing and Prephasing
A Read with a base that is phasing
B Read with a base that is prephasing.
RTA2 corrects the effects of phasing and prephasing, which maximizes the data quality at every cycle
throughout the run.
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
500 uses 2-channel sequencing, which requires only 2 images to encode the data for 4 DNAbases, 1 from
the red channel and 1 from the green channel.
Intensities extracted from an image compared to another image result in 4 distinct populations, each
corresponding to a nucleotide. The base calling process determines to which population each cluster
belongs.
Figure 23 Visualization of Cluster Intensities
Document # 15046563 v04
For Research Use Only. Not for use in diagnostic procedures.
40
NextSeq 500 System Guide
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