Real-Time Analysis

The NovaSeq 6000Dx Instrument runs RTA3, an implementation of Real-Time Analysis software, on the instrument Compute Engine (CE). RTA3 extracts intensities from images received from the camera, performs base calling, assigns a quality score to base calls, aligns to PhiX, and reports data in InterOp files.

To optimize processing time, RTA3 stores information in memory. If RTA3 is terminated, processing does not resume and any run data being processed in memory is lost.

RTA3 requires tile images contained in local system memory for processing. RTA3 receives run information and commands from the NVOS.

Images for each color channel are passed in memory to RTA3 as tiles. From these images, RTA3 outputs a set of quality-scored base call files and filter files. All other outputs are supporting output files.

File Type

Description

Base call files

Each tile that is analyzed is included in a concatenated base call (*.cbcl) file. Tiles from the same lane and surface are aggregated into one CBCL file for each lane and surface.

Filter files

Each tile produces a filter file (*.filter) that specifies whether a cluster passes filters.

RTA3 provides real-time metrics of run quality stored as InterOp files, which are a binary output containing tile, cycle, and read-level metrics.

RTA3 creates log files and writes them to the Logs folder. Errors are recorded in a text file in *.log file format.

The following log files are transferred to the final output destination at the end of processing:

• info_00000.log summarizes important run events.
• error_00000.log lists errors that occurred during a run.
• warning_00000.log lists warnings that occurred during a run.

Tiles are small imaging areas on the flow cell. The camera takes one image of each swath, which the software divides into tiles for RTA3 processing. The total number of tiles depends on how many lanes, swaths, and surfaces are imaged on the flow cell.

• S2 flow cells have a total of 1408 tiles.
• S4 flow cells have a total of 3744 tiles.

Flow Cell Component

S2

S4

Description

Lanes

2

4

A lane is a physical channel with input and output ports.

Surfaces

2

2

The S2 and S4 flow cells are imaged on two surfaces: the top and bottom. The top surface of a tile is imaged first.

Swaths per lane

4

6

A swath is a column in a flow cell lane that the camera captures as one scanned image.

Tiles per swath

88

78

A tile is a portion of a swath and depicts an imaged area on the flow cell.

Total tiles generated

1408

3744

Lanes × surfaces × swaths × tiles per swath equals the total number of tiles.

The tile name is a five-digit number that represents the tile position on the flow cell. For example, tile name 1_1205 indicates lane 1, top surface, swath 2, tile 5.

• The first digit is the lane number:
– 1 or 2 for an S2 flow cell.
– 1, 2, 3, or 4 for an S4 flow cell.
• The second digit represents the surface: 1 for top or 2 for bottom.
• The third digit represents the swath number:
– 1, 2, 3, or 4 for an S2 flow cell.
– 1, 2, 3, 4, 5, or 6 for an S4 flow cell.
• The last two digits represent the tile number. The numbering starts with 01 at the outlet end of the flow cell through 88 or 78 at the inlet end.
– 01 through 88 for an S2 flow cell.
– 01 through 78 for an S4 flow cell.