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 the sequencing run. The NextSeq 1000/2000 utilizes patterned flow cell technology, which uses clustered fiducials for autocenter.

There is a total of 4 fiducials in the NextSeq 1000/2000 flow cells, two on the top surface and two on the bottom surface, and to pass autocenter, the instrument must locate at least three fiducials. Those fiducials must contain signal from the blue channel (ie, have an A or a C base call at cycle 1.)

The Control Software captures the first autocenter image at a preset X/Y coordinate as indicated in the config file and locates the fiducial based on software calculations after the initial image. This process is repeated for each fiducial.

Viewing Autocenter Images

Autocenter images are .tif files and require a compatible viewer to visualize. ImageJ is the software 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 contains three types of files 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 using the measure feature. Create a shape, select Analyze, and then select Measure.

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