Chemistry and imaging on NextSeq 500/550

Chemistry and base calling

• The NextSeq 500/550 uses two-color chemistry, enabling very fast imaging.

• Signal in only the red channel results in a C call, whereas signal in only the green channel results in a T call.

• Signal in both channels yields an A call, and a G call results from the absence of signal in either channel.

• Because of this, there must be at least one non-G base in the first 5 cycles of sequencing for a cluster to be registered during template generation.

• Additionally, a G overcall in a run is generally indicative of a failure of clustering (or paired-end turnaround) or very low clustering.

Reference:

• Two-Channel SBS Technology

Imaging

Review the section titled 'Flow Cell Tiles' of the NextSeq 500 and NextSeq 550 Sequencing System Guide.

Camera numbering

• The NextSeq 500/550 uses six cameras to image the flow cell.

• Cameras are numbered 1-6. Cameras 1-3 image lane one.

• Cameras 4-6 image lane three.

• After lanes 1 and 3 are imaged, the imaging module moves on the X-axis to image lanes 2 and 4.

  

Lane numbering

• Lanes 1 and 3, called lane pair A, are imaged at the same time.

• Lanes 2 and 4, called lane pair B, are imaged when imaging of lane pair A is complete.

• A Lane Pair A—Lanes 1 and 3.

• B Lane Pair B—Lanes 2 and 4.

  

Swath numbering

• Each lane is imaged in three swaths.

• Swaths are numbered 1-3 for high output flow cells.

  

Tile numbering

• There are 12 tiles in each swath of each camera segment.

• Tiles are numbered 01-12, regardless of swath number or camera segment, and represented in two digits.

• The complete tile number includes 5 digits to represent the location, as follows:

  • Surface—1 represents the top surface; 2 represents the bottom surface.

  • Swath—1, 2, or 3.

  • Camera—1, 2, 3, 4, 5, or 6.

  • Tile—01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, or 12.

Example: Tile number 12508 indicates top surface, swath 2, camera 5, and tile 8.

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