Real-Time Analysis Workflow

Registration

Records the location of each cluster on the patterned flow cell.

 

Intensity extraction

Determines an intensity value for each cluster.

 

Phasing correction

Corrects the effects of phasing and prephasing.

 

Base calling

Determines a base call for every cluster.

 

Quality scoring

Assigns a quality score to every base call.

A quality score (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. After the Q-score is determined, results are recorded in base call (*.cbcl) files.

The Q-score succinctly communicates small error probabilities. Quality scores are represented as Q(X), where X is the score. The following table shows the relationship between a quality score and error probability.

Q-Score Q(X)

Error Probability

Q40

0.0001 (1 in 10,000)

Q30

0.001 (1 in 1000)

Q20

0.01 (1 in 100)

Q10

0.1 (1 in 10)

Quality scoring calculates a set of predictors for each base call, and then uses the predictor values to look up the Q-score in a quality table. Quality tables are created to provide calibrated quality predictions for runs generated by a specific configuration of sequencing platform and version of chemistry.

Quality scoring is based on a modified version of the Phred algorithm.

To generate the Q-table for the NextSeq 1000/2000 Sequencing System, three groups of base calls were determined, based on the clustering of these specific predictive features. Following grouping of the base calls, the mean error rate was empirically calculated for each of the three groups and the corresponding Q-scores were recorded in the Q-table alongside the predictive features correlating to that group. As such, only three Q-scores are possible with RTA and these Q-scores represent the average error rate of the group. Overall, this process results in simplified, yet highly accurate quality scoring. The RFID on the cartridge determines the version of RTA used.

 

Q Table

Base Call Groups

Specific Scores Assigned

RTA Version Used

XLEAP-SBS cartridges (50, 100, 200, 300, and 600-cycle)

Marginal (≤ Q17), medium (Q18-29), and high-quality (≥ Q30)

12, 24, and 40 respectively

RTA4

Standard SBS cartridges (50, 100, 200, and 300-cycle)

Marginal (≤ Q17), medium (Q18-29), and high-quality (≥ Q30)

12, 26, and 34 respectively

RTA3

Standard SBS cartridges (600-cycle)

Marginal (≤ Q17), medium (Q18-29), and high-quality (≥ Q30)

9, 20, and 34 respectively

RTA3

Also, a null score of 0 is assigned to any no-calls. This Q-score reporting model reduces storage space and bandwidth requirements without affecting accuracy or performance.