Sequencing Overview

The following information includes additional details on the MiSeq i100 Plus sequencing workflow.

Cluster Generation

The library is automatically denatured into single strands onboard the instrument. During cluster generation, single DNA molecules are bound to the surface of the flow cell and amplified to form clusters. Cluster generation takes ~2 hours.

Sequencing

Clusters are imaged using two-channel chemistry, one green channel and one blue channel, to encode data for the four nucleotides. The flow cell sensors, comprised of tiles, are imaged at one time. The process is repeated for each cycle of sequencing.

Primary Analysis

Following image analysis, Real-Time Analysis (RTA) 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 MiSeq i100 Plus 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 RTA in real time, use the MiSeq i100 Plus 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.

Secondary Analysis

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 prebuilt ICA pipelines or create custom pipelines using your sequencing and analysis data.

If analyzing sequencing data in the cloud, CBCL data is uploaded automatically to the cloud and is 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.

•

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

•

For more information on DRAGEN Secondary Analysis, refer to the DRAGEN Bio-IT Platform support page.

•

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

•

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