RTA v2 provides real-time metrics of run quality stored as InterOp files. InterOp files are a binary output
containing tile, cycle, and read-level metrics, and are required to view real-time metrics using the Sequencing
Analysis Viewer (SAV). For the latest version of SAV, visit the SAV downloads page on the Illumina website.
Error Handling
RTA2 creates log files and writes them to the RTALogs folder. Errors are recorded in an error file in *.tsv file
format.
The following log and error files are transferred to the final output destination at the end of processing:
u *GlobalLog*.tsv summarizes important run events.
u *LaneNLog*.tsv lists processing events for each lane.
u *Error*.tsv lists errors that occurred during a run.
u *WarningLog*.tsv lists warnings that occurred during a run.
Real-Time Analysis Workflow
Template generation Maps cluster locations.
Registration and intensity
extraction
Records the location of each cluster on the flow cell and 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.
Template Generation
The first step in the RTAworkflow is template generation, which defines the position of each cluster in a tile
using X and Y coordinates.
Template generation requires image data from the first 5 cycles of the run. After the last template cycle for a
tile is imaged, the template is generated.
NOTE
To detect a cluster during template generation, there must be at least 1 base other than G in the first 5
cycles. For any index sequences, RTA v2 requires at least 1 base other than G in the first 2 cycles.
The template is used as a reference for the subsequent step of registration and intensity extraction. Cluster
positions for the entire flow cell are written to cluster location (*.locs) files, 1 file for each lane.
Registration and Intensity Extraction
Registration and intensity extraction begin after template generation.
u Registration aligns images produced over every subsequent cycle of imaging against the template.
u Intensity extraction determines an intensity value for each cluster in the template for a given image.
If registration fails for any images in a cycle, no base calls are generated for that tile in that cycle. Use the
Sequencing Analysis Viewer (SAV)software to examine thumbnail images and identify images that failed
registration.
Document # 15046563 v04
For Research Use Only. Not for use in diagnostic procedures.
39
NextSeq 500 System Guide
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