Introduction
This documentation describes strategies for optimizing cluster density and preventing and diagnosing clustering issues on Illumina flow cells. Use this guide as a reference when preparing and sequencing libraries.
Related Videos
Several topics in this guide have complementary videos. Visit the Training page of the Illumina website to watch the videos.
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Topic |
Video |
|---|---|
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Cluster generation and sequencing by synthesis (SBS) |
Sequencing: Illumina Technology |
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Library quantification |
How do I achieve consistent quantitation? Part 1 How do I achieve consistent quantitation? Part 2 |
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Base calling, clusters passing filter, and nucleotide diversity |
How do I optimize amplicon sequencing data? Part 1 How do I optimize amplicon sequencing data? Part 2 |
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Overclustering nonpatterned flow cells |
Is my HiSeq or MiSeq run overclustered? |
Clustering Overview
A cluster is a clonal group of library fragments on a flow cell. Each cluster produces one single read or one paired-end read. For example, a flow cell with 10,000 clusters produces 10,000 single reads or 20,000 paired-end reads.
A paired-end read sequences both ends of a DNA fragment in the same run, while a single read sequences only one end. For more information, see Indexed Sequencing on Illumina Systems.
During clustering, each fragment binds to the flow cell and seeds a template that is amplified until the cluster consists of hundreds or thousands of copies. The number of clusters and the location of each cluster is fixed throughout a run. An incorporation mix flows through the flow cell, tagging each fragment with a fluorescent‑labeled nucleotide. Base calls are made from the resulting signal (intensity) that each cluster emits.
Base Calling and Passing Filter
The Real-Time Analysis software runs on the instrument control computer. During a sequencing run, it extracts intensities from images to perform base calling, and then assigns a quality score to the base call.
Cluster density on a flow cell impacts the following steps in the Real-Time Analysis workflow:
| • | Passing filter—During cycles 1–25 of Read 1, a filter removes unreliable clusters from the image extraction results. Clusters pass filter when certain quality specifications are met. For more information, see Calculating Percent Passing Filter for Patterned and Nonpatterned Flow Cells (Pub. No. 770-2014-043). |
| • | Registration and intensity extraction—For each cluster on the flow cell, the software records a cluster location and calculates an intensity value. |
| • | Template generation—The software analyzes images from the first 5–7 cycles of a run to map the location of each cluster on a nonpatterned flow cell. (Cluster locations on a patterned flow cell are predetermined.) The resulting template is input for the subsequent registration step. |
The implementation of Real-Time Analysis, including workflow steps, varies by system. For system-specific information, see the system guide for your instrument.
