Lecture 3

Illumina sequencing
technology
Illumina sequencing technology: Introduction

• Relies on bridge PCR or bridge amplification

• The single molecule amplification step for the Illumina
Genome Analyzer starts with an Illumina-specific adapter
library
• Takes place on the oligo-derivatized surface of a flow cell
• Performed by an automated device called a Cluster Station
 DNA library preparation
• DNA is fragmented and the fragmented DNA is used for the
preparation of DNA library
• Specialized adapters are added to DNA fragment ends
• DNA libraries can be constructed by any method that gives rise to a
mixture of adaptor-flanked fragments
• Primers (with sequences complementary to adapters) are coated on
a solid substrate, attached at their 5’ ends inside of the flow cell
channels
• Library is loaded into a flow cell and the fragments hybridize to the
flow cell surface
• Each bound fragment is amplified into a clonal cluster through
bridge amplification
• The flow cell is an 8-channel sealed acrylamide-coated glass
microfabricated device that allows bridge amplification of fragments
on its surface
 Sequence by Synthesis
• SBS uses four fluorescently labeled
nucleotides. During each sequencing cycle a
single labeled dNTP is added to the nucleic
acid chain. The nucleotide label serves as a
terminator for polymerization, so after each
dNTP incorporation, the fluorescent dye is
imaged to identify the base and then
enzymatically cleaved to allow incorporation
of the next nucleotide.
 Sequence by Synthesis
• At the end of bridge amplification, all of the reverse strands are
washed off the flow cell, leaving only forward strands. Primers
attach to the forward strands and add fluorescently tagged
nucleotides to the DNA strand. Only one base is added per round. A
reversible terminator is on every nucleotide to prevent multiple
additions in one round. Using the four-colour chemistry, each of the
four bases has a unique emission, and after each round, the
machine records which base was added. Starting with the launch of
the NextSeq and later the MiniSeq, Illumina introduced a new two-
colour sequencing chemistry. Nucleotides are distinguished by
either one of two colours (red or green), no colour ("black") or
binding both colours (appreading orange as a mixture between red
and green).
 Flowchart

• DNA fragmentation
• Anneal platform-specific linkers to the ends of
DNA fragments
• Cluster generation: bridge amplification
• Sequencing by synthesis
• Data analysis
Thank
you