Illumina Sequencing Workflow

Illumina sequencing involves four key steps:

1. Sample Preparation

   • Various sample preparation methods exist.
   • All methods add adaptors to the ends of DNA fragments.
   • Reduced cycle amplification introduces motifs such as:
     • Sequencing binding site.
     • Indices.
     • Regions complementary to flow cell oligos.

2. Cluster Generation

   • Clustering: Each fragment molecule is isothermally amplified.
   • Flow Cell: A glass slide with lanes, each lane as a channel coated with a lawn of two types of oligos.
     • First oligo enables hybridization with adapter region on fragment strands.
     • Polymerase creates complementary strand, forming double-stranded molecule.
     • Original strand denatured and washed away.
     • Bridge Amplification:
       • Strand folds over, hybridizes with second oligo.
       • Polymerases generate a complementary strand creating a double-stranded bridge.
       • Bridge denatured, resulting in two copies tethered to flow cell.
     • Repeated for millions of clusters, achieving clonal amplification.
   • Post-Amplification:
     • Reverse strands cleaved and washed off.
     • Three prime ends blocked to prevent unwanted priming.

3. Sequencing

   • Begins with extension of the first sequencing primer producing the first read.
   • Sequencing-by-Synthesis:
     • Fluorescently tagged nucleotides are incorporated one at a time.
     • Clusters excited by light, emitting fluorescent signals.
     • Number of cycles determines read length.
     • Wavelength and signal intensity determine base call.
   • Massively parallel process sequencing hundreds of millions of clusters.
   • Post-Read Steps:
     • Read product washed away.
     • Index 1 read primer introduced and hybridized.
     • After index read, product washed off, three prime ends deprotected.
     • Template folds and binds second oligo on flow cell.
     • Index 2 read in similar fashion as index 1.
     • Double-stranded bridge formed, linearized, ends blocked.
     • Original forward strand cleaved and washed away.
     • Read 2 sequencing begins with read 2 primer introduction.
     • Sequencing steps repeated for desired read length.
     • Read 2 product washed away.

4. Data Analysis

   • Following sequencing, data is analyzed to interpret and derive meaningful results from the sequencing data collected.