Download Directed Electronics equalizer 2500 User guide

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 2500 using Real-Time Analysis 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 if 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. The percentage of
clusters passing filter is represented in analysis reports as %PF.
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.
Quality Scoring
A quality score, or Q-score, is a prediction of the probability of an incorrect base call. A
higher Q-score implies that a base call is more reliable and more likely to be correct.
The Q-score serves as a compact way to communicate small error probabilities. Quality
scores are represented as Q(X), where X is the score. The following table shows the
relationship between the quality score and error probability.
Q-Score Q(X)
Q40
Q30
Q20
Q10
HiSeq 2500 System User Guide
Error Probability
0.0001 (1 in 10,000)
0.001 (1 in 1,000)
0.01 (1 in 100)
0.1 (1 in 10)
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Real-Time Analysis Workflow
Base Calling