Sequencing Overview

During cluster generation, single DNA molecules are bound to the surface of the flow cellA glass slide with physically separated lanes. The lanes are coated with oligos complementary to the library adapter sequences, allowing the library to adhere for a sequencing run. and simultaneously amplified to form clusters.

Clusters are imaged using two-channel chemistry, one green channel and one blue channel, to encode data for the four nucleotides. After one tile on the flow cell is imaged, the next tile is imaged. The process is repeated for each cycle of sequencing (~5 minutes per cycle).

Following image analysis, Real-Time Analysis (RTA4) software performs base callingDetermining a base (A, C, G, or T) for every cluster in a tile at a specific cycle., filtering, and quality scoringCalculates a set of quality predictors for each base call, and then uses the predictor value to look up the Q-score.. As the run progresses, the NovaSeq X Series Control Software automatically transfers base call filesContains the base call and associated quality score for every cluster of each sequencing cycle. (*.CBCL) to the specified output location for data analysis. To view quality metrics generated by RTA4 in real-time, use the NovaSeq X Series Control Software, Sequencing Analysis Viewer (SAV), or BaseSpace Sequence Hub.

Secondary analysis begins after sequencing is complete. The method of secondary data analysis depends on your application and system configuration.

BaseSpace Sequence Hub and Illumina Connected Analytics (ICA) are the Illumina cloud computing environments for data analysis, storage, and run monitoring. Run monitoring is only visible in BaseSpace Sequence Hub. BaseSpace Sequence Hub hosts DRAGEN and BaseSpace Sequence Hub apps, which support common analysis methods for sequencing. Illumina Connected Analytics hosts the DRAGEN for ICA pipelines. You can use pre-built ICA pipelines or create custom pipelines using your sequencing and analysis data.

If analyzing sequencing data in the cloud, CBCL data are uploaded automatically to the cloud and are available in BaseSpace Sequence Hub and ICA. Analysis automatically begins after the data upload completes.

If analyzing sequencing data locally, DRAGEN secondary analysis is performed on-instrument and output files are stored in a selected output folder.

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For more information on BaseSpace Sequence Hub, refer to BaseSpace Sequence Hub support page.

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For more information on Illumina DRAGEN Bio-IT Platform, refer to Illumina DRAGEN Bio-IT Platform support page.

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For more information on Illumina Connected Analytics, refer to Illumina Connected Analytics support page.

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For an overview of all apps, refer to BaseSpace Sequence Hub Apps.