Real-Time Analysis Overview

The NextSeq 1000/2000 Sequencing System runs Real-Time Analysis (RTA) software, on the instrument Compute Engine (CE). RTA 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 for viewing in the instrument control software.

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

RTA requires tile images contained in local system memory for processing. RTA receives run information and commands from the control software.

Images for each color channel are passed in memory to RTA as tiles. From these images, RTA 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 1 *.cbcl file for each lane and surface.

Filter files

Each tile produces a filter file (*.filter) that 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 run.

Output files are used for downstream analysis in DRAGEN and BaseSpace Sequence Hub.

RTA 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 per tile.

Flow Cell Tiles

Flow Cell

Lanes

Surfaces

Swaths per lane

Tiles per swath

Total tiles generated

P1 Flow Cell

1

2

4

4

32

P2 Flow Cell (100-cycle, 200-cycle, 300-cycle kits)

1

2

6

11

132

P2 XLEAP-SBS Flow Cell (600-cycle kits)

1

2

4

14

112

P2 Standard SBS Flow Cell (600-cycle kits)

1

2

4

12

96

P3 Flow Cell

2

2

6

14

336

P4 Flow Cell

2

2

6

16

384

Descriptions

• Lanes—Lanes are optically distinct, but not fluidically separate channels.
• Surfaces—The flow cells are imaged on two surfaces: the top and bottom. The top surface of a tile is imaged first.
• Swaths per lane—A swath is a column in a flow cell lane.
• Tiles per swath—A tile is a portion of a swath and depicts an imaged area on the flow cell.
• Total tiles generated—Lanes × surfaces × swaths × tiles per swath equals the total number of tiles.

Tile Naming

• The tile name is a four-digit number that represents the tile position on the flow cell. For example, tile name 1205 indicates top surface, swath 2, tile 05.
• The first digit represents the surface: 1 for top or 2 for bottom.
• The second digit represents the swath number: 1, 2, 3, 4, 5, or 6.
• The last two digits represent the tile number.