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Real-Time Analysis Workflow

HiSeq 2500 System User Guide

119

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 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) Error Probability

Q40 0.0001 (1 in 10,000)

Q30 0.001 (1 in 1,000)

Q20 0.01 (1 in 100)

Q10 0.1 (1 in 10)

1 2 ... 124 125 126 127 128 129 130 131 132 133 134 ... 153 154

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Directed Electronics equalizer 2500 Betriebsanweisung Seite 129