Color Matrix Correction
After registration and intensity extraction, RTA corrects for cross talk between channels. Cross talk occurs
when a cluster shows intensity in the C channel and some intensity also shows in the A channel, for example.
Using a 4 x 4 color matrix, RTA generates matrix-corrected intensities with reduced or no cross talk, and
balances differences in overall intensity between color channels.
Empirical 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 20
Phasing and Prephasing
A
Read with a base that is phasing
B
Read with a base that is prephasing.
RTA corrects the effects of phasing and prephasing using the empirical phasing correction algorithm, which
maximizes the data quality at every cycle throughout the run.
Base Calling
After raw intensities are corrected for cross talk, phasing, and prephasing, the channel with the brightest
intensity is the call for that cluster in that cycle. Base calling on the HiSeq 2000 using RTA begins after cycle
12.
Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. Base calls are
saved to base call (*.bcl) files, which are binary files with 1 byte per call and quality score. Each base call file
contains the base call and the base call quality score. To make a base call, clusters must first pass the
chastity filter. Clusters that do not pass filter or cannot be called because they are off-image or failed image
registration are labeled no-calls. No-calls are represented as (N).
Clusters Passing Filter
During the first 25 cycles of Read 1, the chastity filter removes the least reliable clusters from analysis results.
Clusters pass filter when no more than 2 base calls have a chastity value below 0.6 in the first 25 cycles.
Chastity is the ratio of the brightest base intensity divided by the sum of the brightest and the second
brightest base intensities. Analysis reports represent the percentage of clusters passing filter as %PF.
Low quality clusters are removed from the raw cluster count during template generation, which yields a
relatively high percentage of clusters passing filter.
Document # 15011190 v03
For Research Use Only. Not for use in diagnostic procedures.
58
HiSeq 2000 System Guide