Illumina Sequencing Technology

Technology Spotlight: Illumina

Sequencing

Introduction

Illumina sequencing technology leverages clonal array formation and

proprietary reversible terminator technology for rapid and accurate

large-scale sequencing. The innovative and exible sequencing system

enables a broad array of applications in genomics, transcriptomics,

and epigenomics.

Cluster Generation

Sequencing templates are immobilized on a proprietary ow cell

surface (Figure 1) designed to present the DNA in a manner that

facilitates access to enzymes while ensuring high stability of surface-

bound template and low non-specic binding of uorescently labeled

nucleotides. Solid-phase amplication (Figures 2–7) creates up to

1,000 identical copies of each single template molecule in close prox-

imity (diameter of one micron or less). Because this process does not

involve photolithography, mechanical spotting, or positioning of beads

into wells, densities on the order of ten million single-molecule clusters

per square centimeter are achieved.

Sequencing by Synthesis

Sequencing by synthesis (SBS) technology uses four uorescently-

labeled nucleotides to sequence the tens of millions of clusters on

the ow cell surface in parallel (Figure 8–12). During each sequencing

cycle, a single labeled deoxynucleoside triphosphate (dNTP) is added

to the nucleic acid chain. The nucleotide label serves as a terminator

for polymerization, so after each dNTP incorporation, the uorescent

dye is imaged to identify the base and then enzymatically cleaved to

allow incorporation of the next nucleotide. Since all four reversible

terminator-bound dNTPs (A, C, T, G) are present as single, separate

molecules, natural competition minimizes incorporation bias. Base

calls are made directly from signal intensity measurements during each

cycle, which greatly reduces raw error rates compared to other tech-

nologies. The end result is highly accurate base-by-base sequencing

that eliminates sequence-context specic errors, enabling robust base

calling across the genome, including repetitive sequence regions and

within homopolymers.

Analysis Pipeline

The Illumina sequencing approach is built around a massive quantity

of sequence reads in parallel. Deep sampling and uniform cover-

age is used to generate a consensus and ensure high condence in

determination of genetic differences. Deep sampling allows the use of

weighted majority voting and statistical analysis, similar to conventional

methods, to identify homozygotes and heterozygotes and to distin-

guish sequencing errors. Each raw read base has an assigned quality

score so that the software can apply a weighting factor in calling differ-

ences and generating condence scores.

Data Collection, Processing, and Analysis

Illumina data collection software enables users to align sequences

to a reference in resequencing applications (Figure 13). Developed

in collaboration with leading researchers, this software suite includes

the full range of data collection, processing, and analysis modules to

streamline collection and analysis of data with minimal user interven-

tion. The open format of the software allows easy access to data at

various stages of processing and analysis using simple application

program interfaces.

Data Accuracy and Workow Simplicity

The TruSeq family of reagents represents the latest advancement of

Illumina’s sequencing by synthesis (SBS) technology. From sample

prep through DNA sequencing, TruSeq reagent chemistry enables

Illumina sequencing to provide the most accurate data across a broad

range of applications. With highest yield of error-free reads and most

base calls above Q30, researchers can have the highest condence in

their data integrity to draw sound biological conclusions.

A highly automated, streamlined workow requires minimal instrument

hands-on time. With the ability to generate several gigabases of DNA

sequence per run, even large mammalian genomes can be sequenced

in weeks rather than years. The capacity to accommodate many

samples per ow cell means that runs can be tailored to the demands

of diverse applications.

Illumina Sequencing Technology

Highest data accuracy, simple workow, and a broad range of applications.

Figure 1: Illumina Flow Cell

Several samples can be loaded onto the eight-lane ow cell for simultane-

ous analysis on an Illumina Sequencing System.

Technology Spotlight: Illumina

Sequencing

Figure 2: Prepare Genomic DNA Sample

Randomly fragment genomic DNA and ligate adapters to both ends of the

fragments.

Figure 3: Attach DNA to Surface

Bind single-stranded fragments randomly to the inside surface of the ow

cell channels.

Figure 5: Fragments Become Double Stranded

The enzyme incorporates nucleotides to build double-stranded bridges on

the solid-phase substrate.

Adapters

DNA

Adapter

DNA

fragment

Dense lawn

of primers

Adapter

Figure 4: Bridge Amplication

Add unlabeled nucleotides and enzyme to initiate solid-phase bridge

amplication.

Attached

terminus

Attached

terminus

Free

terminus

Technology Spotlight: Illumina

Sequencing

Figure 6: Denature the Double-Standed Molecules

Denaturation leaves single-stranded templates anchored to the substrate.

Figure 7: Complete Amplication

Several million dense clusters of double-stranded DNA are generated in

each channel of the ow cell.

Figure 8: Determine First Base

The rst sequencing cycle begins by adding four labeled reversible

terminators, primers, and DNA polymerase.

Attached

Clusters

Laser

Figure 9: Image First Base

After laser excitation, the emitted uorescence from each cluster is captured

and the rst base is identied.

Technology Spotlight: Illumina

Sequencing

Figure 10: Determine Second Base

The next cycle repeats the incorporation of four labeled reversible

terminators, primers, and DNA polymerase.

Figure 12: Sequencing Over Multiple Chemistry Cycles

The sequencing cycles are repeated to determine the sequence of bases in

a fragment, one base at a time.

Figure 11: Image Second Chemistry Cycle

After laser excitation, the image is captured as before, and the identity of

the second base is recorded.

Figure 13: Align Data

The data are aligned and compared to a reference, and sequencing

differences are identied.

Laser

GCTGA...

Technology Spotlight: Illumina

Sequencing

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Pub. No. 770-2007-002 Current as of 11 October 2010

Additional Information

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