Autocenter process and images on the NextSeq 1000/2000

Background: Autocenter is the process of finding the initial focus after clustering has completed, just prior to the start of sequencing. The NextSeq 1000/2000 utilizes patterned flow cell technology, which uses clustered fiducials for autocenter.

The NextSeq 1000/2000 flow cells have a total of 4 fiducials, two on the top surface and two on the bottom. Autocenter is performed in the blue channel only.

To pass autocenter, the NextSeq 1000/2000 must locate three of the four fiducials. Those fiducials must contain signal from the blue channel, ie, contain an A or a C base call at cycle 1.

The NextSeq 1000/2000 control software software will capture the first autocenter image at a preset X/Y coordinate as indicated in the config file and will locate the fiducial based on software calculations after the initial image. This is repeated for each fiducial.

Viewing Autocenter Images Autocenter images are .tif files and require a compatible viewer to visualize. ImageJ is recommended and can be downloaded here.

Autocenter images are found in the NextSeq 1000/2000 run folder at: /usr/local/Illumina/runs/[runfolder]/AutoCenter

This folder will contain three types of files types related to the autocenter images on the NextSeq 1000/2000.

1. AutoCenterFiducialFinder_fiducial_X*[value]_Y[value]*_CorrelationBased.tif

• This image is the best fit calculation of the two RAW_stack images from each fiducial. In successful runs, expect one per fiducial, total 4 per run.

1. [YEAR-MONTH-DATE-TIME]_RAW_stack.tif

• These are the initial autocenter images captured. In successful runs, expect a minimum of two RAW_stack.tif images for each fiducial, total 8 per run.

1. [YEAR-MONTH-DATE-TIME]_AutoCenterData.csv

• This file shows the expected location, the actual location and difference between the locations per fiducial. It details the offset calculations for both surfaces.

Autocenter fiducial intensity can be measured by hovering the cursor over a region of the fiducial and viewing the value found in the ImageJ toolbar.

Intensity can also be measured over a larger area with the measure feature. Create a shape, select Analyze, select Measure.

For any feedback or questions regarding this article (Illumina Knowledge Article #2724), contact Illumina Technical Support [e-mail].