illumina NextSeq 550Dx Instrument Reference Manual page 61

NextSeq 550Dx Research Mode Instrument Reference Guide
Table 1 Base Calls in 2-Channel Sequencing
Base Red Channel
A 1 (on)
C 1 (on)
G 0 (off)
T 0 (off)
Clusters Passing Filter
During the run, RTA2 filters raw data to remove reads that do not meet the data quality threshold.
Overlapping and low-quality clusters are removed.
For 2-channel analysis, RTA2 uses a population-based system to determine the chastity of a base call.
Clusters pass filter (PF) when no more than 1 base call in the first 25 cycles has a chastity of < 0.63.
Clusters that do not pass filter are not base called.
Indexing Considerations
The process for base calling index reads differs from base calling during other reads.
Index reads must begin with at least 1 base other than G in either of the first 2 cycles. If an Index Read
begins with 2 base calls of G, no signal intensity is generated. Signal must be present in either of the
first 2 cycles to ensure demultiplexing performance.
To increase demultiplexing robustness, select index sequences that provide signal in at least 1 channel,
preferably both channels, for every cycle. Following this guideline avoids index combinations that result
in only G bases at any cycle.
Red channel—A or C
u
Green channel—A or T
u
This base calling process ensures accuracy when analyzing low-plex samples.
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 higher quality and more likely to be correct.
The Q-score is 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)
NOTE
Quality scoring is based on a modified version of the Phred algorithm.
Document # 1000000041922 v01
For Research Use Only. Not for use in diagnostic procedures.
Green
Result
Channel
1 (on) Clusters that show intensity in both the red and green channels.
0 (off) Clusters that show intensity in the red channel only.
0 (off) Clusters that show no intensity at a known cluster location.
1 (on) Clusters that show intensity in the green channel only.
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