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Overview of Illumina Sequencing Process

Mar 13, 2025

Illumina Sequencing Workflow

The Illumina sequencing workflow consists of four key steps: sample preparation, cluster generation, sequencing, and data analysis. Each step is crucial to the successful sequencing of DNA fragments.

1. Sample Preparation

  • Adaptors Addition: Adaptors are added to the ends of DNA fragments.
  • Reduced Cycle Amplification: Introduces additional motifs such as sequencing binding sites, indices, and regions complementary to flow cell oligos.

2. Cluster Generation

  • Isothermal Amplification: Each fragment molecule is isothermally amplified.
  • Flow Cell: A glass slide with lanes, each coated with a lawn of two types of oligos.
    • Hybridization: Enabled by the first type of oligo complementary to the adapter region.
    • Polymerase Action: Creates a complement of the hybridized fragment.
    • Bridge Amplification: The strand folds and hybridizes to the second type of oligo, generating a double-stranded bridge. This bridge is denatured into single-stranded copies tethered to the flow cell.
  • Clonal Amplification: Repeated over millions of clusters, resulting in clonal amplification.
  • Cleaving and Blocking: Reverse strands are cleaved and washed off, with the three-prime ends blocked.

3. Sequencing

  • Sequencing Primer Extension: Begins with the first sequencing primer to produce the first read.
  • Sequencing-by-Synthesis: In each cycle, fluorescently tagged nucleotides are added to the growing chain, excited by a light source, emitting a characteristic fluorescent signal.
    • Cycle Count: Determines the read length.
    • Emission Wavelength and Signal Intensity: Determine the base call.
  • Parallel Processing: Hundreds of millions of clusters sequenced simultaneously.
  • Index Reading:
    • Index 1: After the first read, the index 1 read primer is introduced and hybridized.
    • Index 2: After index 1, the template folds over for index 2 reading.
  • Read 2 Sequencing: After linearization and blocking, read 2 begins with a new sequencing primer.

Summary

  • The sequencing process consists of multiple cycles ensuring accurate and long reads.
  • Massively Parallel Process: Ensures high throughput sequencing of hundreds of millions of clusters.
  • Final Steps: Include deprotection of the 3' ends and washing away of products to prepare for subsequent reads.

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