# 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](https://www.illumina.com/science/technology/next-generation-sequencing/sequencing-technology/2-channel-sbs.html)

**Imaging**

Review the section titled 'Flow Cell Tiles' of the [NextSeq 500 and NextSeq 550 Sequencing System Guide](https://support.illumina.com/downloads/nextseq-500-550-systems-product-documentation.html).

**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.

  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-5c1a53ad5fe00fe4674829960ac8a94b1ac9540d%2Fimage1.png?alt=media\&token=316f3c76-d312-4861-a347-0c3208985266)

**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.

  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-41c399d77562a5f7e5fe44d4b8e040a7934ed14d%2Fimage2.png?alt=media\&token=a7691f46-d0e3-42bd-a87d-7fe2a6f3ff33)

**Swath numbering**

* Each lane is imaged in three swaths.
* Swaths are numbered 1-3 for high output flow cells.

  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-d58f95dca7fc8a48c57d445a68c990d862c4e9ea%2Fimage3.png?alt=media)

**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.

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-043fe36d514ad73035a5e149174f49c7a9addc17%2Fimage4.png?alt=media)

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                        |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #2078), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002078%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |