Appendix B Real-Time Analysis
Real-Time Analysis Overview 38
Real-Time Analysis Workflow 39
Sequencing Output Files 42
Flow Cell Tiles 42
Output Folder Structure 45
Real-Time Analysis Overview
The NextSeq 500 uses an implementation of Real-Time Analysis (RTA) software called RTA2. RTA2 runs on
the instrument computer and extracts intensities from images, performs base calling, and assigns a quality
score to the base call. RTA2 and the control software communicate through a web HTTP interface and
shared memory files. If RTA2 is terminated, processing does not resume and run data are not saved.
NOTE
Demultiplex performance is not calculated. Therefore, the Index tab in Sequencing Analysis Viewer (SAV)is
not populated.
RTA2 Inputs
RTA2 requires the following input for processing:
u Tile images contained in local system memory.
u RunInfo.xml, which is generated automatically at the beginning of the run and provides the run name,
number of cycles, whether a read is indexed, and the number of tiles on the flow cell.
u RTA.exe.config, which is a software configuration file in XML format.
RTA2 receives commands from the control software about the location of RunInfo.xml and whether an
optional output folder is specified.
RTA v2 Output Files
Images for each channel are passed in memory as tiles. Tiles are small imaging areas on the flow cell defined
as the field of view by the camera. From these images, the software produces output as a set of quality-
scored base call files and filter files. All other files are supporting output files.
File Type Description
Base call files Each tile that is analyzed is included in an aggregated base call (*.bcl) file for each lane and for
each cycle. The aggregated base call file contains the base call and associated quality score for
every cluster in that lane.
Filter files Each tile produces filter information that is aggregated into 1 filter (*.filter) file for each lane. The
filter file specifies whether a cluster passes filters.
Cluster location files Cluster location (*.locs) files contain the X,Y coordinates for every cluster in a tile. A cluster
location file is generated for each lane during template generation.
Base call index files A base call index (*.bci) file is produced for each lane to preserve the original tile information. The
index file contains a pair of values for each tile, which are tile number and number of clusters for
that tile.
Output files are used for downstream analysis in BaseSpace. Alternatively, use bcl2fastq conversion software
for FASTQ conversion and third-party analysis solutions. NextSeq files require bcl2fastq v2.0, or later. For the
latest version of bcl2fastq, visit the NextSeq downloads page on the Illumina website.
Document # 15046563 v04
For Research Use Only. Not for use in diagnostic procedures.
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