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Real-Time Analysis Workflow

HiSeq 2500 System User Guide

117

Real-Time Analysis Workflow

Real-Time Analysis and the control software perform the Real-Time Analysis workflow.

The workflow includes the following steps:

} Template generation—Maps cluster locations.

} Registration and intensity extraction—Records the location of each image on the flow

cell and determines an intensity value for each cluster.

} Color matrix correction—Corrects cross talk between channels.

} Empirical phasing correction—Corrects the effects of phasing and prephasing.

} Base calling—Determines a base call for every cluster.

} Quality scoring—Assigns a quality score to every base call.

Template Generation

The first step of the workflow is template generation, which defines the position of each

cluster in a tile using X and Y coordinates. The template is used as a reference for the

subsequent step of registration and intensity extraction.

Because of the random array of clusters on the flow cell, template generation requires image

data from the first 5 cycles of the run. After the last template cycle for a tile is imaged, the

template is generated.

Cluster positions are written to 1 cluster location (*.locs) file or compressed cluster location

(*.clocs) file for each tile. For more information, see Sequencing Output Files on page 121.

Registration and Intensity Extraction

Registration and intensity extraction begin after the template of cluster positions is

generated.

} Registration aligns the images produced over every cycle subsequent to template

generation against the template.

} Intensity extraction determines an intensity value for each cluster in the template for a

given image.

If registration fails for any images in a cycle, no base calls are generated for that tile in that

cycle. Use the Sequencing Analysis Viewer to examine thumbnail images and identify

images that failed registration. Use the offsets files for troubleshooting registration issues.

For more information, see Sequencing Output Files on page 121.

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Directed Electronics equalizer 2500 Guía de usuario Pagina 127