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HiSeq X System User Guide
57
Base Calling
After raw intensities have been color corrected and phasing and prephasing corrected, the
color channel with the brightest intensity is the base call for that cluster in that cycle. Base
calling on the HiSeq X using RTA v2 begins after cycle 3.
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 if no more than 2 base calls have a chastity value
below 0.6 in the first 25 cycles. Chastity is defined as the ratio of the brightest base intensity
divided by the sum of the brightest and the second brightest base intensities. The
percentage of clusters passing filter is represented in analysis reports as %PF.
The HiSeq X patterned flow cell generates a lower percentage of clusters passing filter
relative to other HiSeq models. The difference is due to the characteristics of an ordered
array of clusters on a patterned flow cell compared to the random array of clusters on a
non-patterned flow cell.
}
Random clusters on a non-patterned flow cell
—Clusters are formed in a random array
and located during template generation. Low quality clusters are removed from the raw
cluster count during template generation, which yields a relatively high percentage of
clusters passing filter.
}
Ordered array of clusters on a patterned flow cell
—Clusters are formed in a
predefined array of nano-wells. Empty wells with no clusters and polyclonal wells
where more than 1 sequence exists are included in the raw cluster count, but do not
pass filter. Therefore, the ordered array on a patterned flow cell yields a relatively low
percentage of clusters passing filter.