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All NGS questions

Question 10 of 20 Fundamentals Concept

How does Illumina sequencing by synthesis read a DNA fragment?

Short answer

Each fragment is copied in place on a flow cell into a cluster of identical molecules. In every cycle a polymerase adds one fluorescently labeled nucleotide with a reversible terminator, a camera images the color, and the dye and block are removed before the next base.

Why

Library fragments carry adapters that let them bind to oligonucleotides on the flow cell surface. Bentley and colleagues describe isothermal bridge amplification, in which each fragment bends over to prime from a neighboring oligonucleotide and is copied repeatedly until it forms a cluster about one micron across. A cluster is bright enough to image, where a single molecule would not be.

Sequencing then proceeds one base per cycle. All four nucleotides are present, each with its own dye and a reversible blocking group that stops synthesis after one addition. Illumina's technical note explains that having all four present as separate molecules lets them compete naturally, which minimizes incorporation bias.

After imaging, the dye and the block are cleaved and the next cycle begins. For paired reads, the template is copied back and the other end is read. The method has limits that show in data.

Base quality is usually highest at the start of a read and degrades toward the 3' end, which is why trimming tools such as cutadapt trim low-quality read ends.

A strong answer covers

  • Bridge amplification makes clonal clusters on the flow cell
  • Reversible terminators allow exactly one base per cycle
  • All four labeled nucleotides compete in each cycle
  • Paired reads come from reading the complementary strand
  • Quality usually falls toward the 3' end of reads

Common mistakes

  • Describing Illumina sequencing as reading single molecules
  • Confusing sequencing by synthesis with Sanger chain termination
  • Assuming quality is uniform along the whole read