Illumina DNA Sequencing

Illumina sequencing workflow

Composed of four basic steps: sample prep, cluster generation, sequencing, and data analysis.

Step 1: Sample preparation

DNA samples are prepared by fragmenting and adding adaptors to the ends of DNA fragments.

Adaptors

Short synthetic sequences ligated to DNA fragments; they allow binding to flow cell oligos, sequencing primer binding, and indexing.

Reduced-cycle amplification

A step during sample prep where additional motifs are added: sequencing binding sites, indices (barcodes), and flow cell–complementary regions.

Step 2: Cluster generation

Each fragment molecule is isothermally amplified on the flow cell, creating clusters of identical sequences.

Flow cell

A glass slide with lanes (channels) coated with a lawn of two types of oligos used for hybridization and amplification.

Flow cell oligos (type 1)

First type of oligo; complementary to the adapter region of one strand of the DNA fragment, enabling hybridization.

Flow cell oligos (type 2)

Second type of oligo; binds the adapter region during bridge amplification, allowing strand folding and copying.

Hybridization in flow cell

DNA fragments hybridize to the first type of oligo via complementarity to their adaptors.

Polymerase action in cluster generation

DNA polymerase extends from the hybridized fragment to synthesize a complementary strand.

Template wash

After initial synthesis, the double-stranded molecule is denatured and the original template strand is washed away.

Bridge amplification

Process where a single-stranded fragment folds over, hybridizes to the second oligo, and is copied, forming a double-stranded “bridge.”

Bridge denaturation

The double-stranded bridge is denatured, leaving two tethered single-stranded molecules.

Clonal amplification

Repeated cycles of bridge amplification produce millions of identical copies of each fragment in a cluster.

Clusters

Groups of clonally amplified identical DNA fragments tethered to the flow cell; millions form in parallel.

Strand selection

After amplification, reverse strands are cleaved and washed off, leaving only forward strands tethered.

3′ end blocking

Three-prime ends of forward strands are chemically blocked to prevent unwanted priming.

Step 3: Sequencing-by-Synthesis (SBS)

Illumina’s proprietary method where fluorescently tagged nucleotides are added one at a time to growing strands and detected.

Sequencing primer

Binds to the adaptor region and initiates synthesis during sequencing.

Cycle of sequencing

In each cycle, fluorescently tagged nucleotides compete for incorporation into the growing DNA strand.

Nucleotide incorporation

Only one nucleotide is incorporated, determined by the sequence of the template.

Fluorescent detection

After each incorporation, clusters are excited by a light source, emitting a characteristic fluorescent signal.

Base calling

The emission wavelength and signal intensity identify which base was added at each cycle.

Read length

Determined by the number of sequencing cycles performed.

Cluster sequencing

All identical strands within a cluster are read simultaneously, ensuring strong signals.

Massively parallel sequencing

Hundreds of millions of clusters are sequenced simultaneously across the flow cell.

Image of flow cell

Each sequencing run captures signals from millions of clusters, though each image shows only a small fraction.

Read 1

The first sequencing read, generated from the forward strand using a sequencing primer.

Read product wash

After each read is completed, the synthesized product is washed away to prepare for the next read step.

Index 1 read

A primer binds to the adaptor; sequencing occurs similar to read 1, producing the first index (barcode).

Index 1 wash

After sequencing, the index read product is washed off; 3′ ends of templates are deprotected.

Template folding (for Index 2)

The strand folds over and binds to the second oligo on the flow cell to enable index 2 reading.

Index 2 read

Sequenced in the same manner as index 1, producing the second barcode.

Bridge reformation

Polymerase extends the second flow cell oligo, creating a double-stranded bridge again.

Linearization after Index 2

The double-stranded DNA is linearized; 3′ ends are blocked again to control priming.

Forward strand cleavage

The original forward strand is cleaved off and washed away, leaving only the reverse strand.

Read 2

Begins with the introduction of a new sequencing primer; sequencing proceeds as in read 1 until desired read length is reached.

Read 2 wash

After sequencing is complete, the second read product is washed away.

Step 4: Data analysis

Computational step where fluorescence data is converted into sequence reads, aligned, and analyzed.