The Illumina sequencing workflow is composed of 4 basic steps– sample prep, cluster generation,
sequencing and data analysis.
There are a number of different ways to
prepare samples.
All preparation methods
add adaptors to the ends of the DNA
fragments.
Through reduced cycle
amplification, additional motifs are
introduced, such as the sequencing binding
site, indices and regions complementary to
the flow cell oligos.
Clustering is a process where each fragment
molecule is isothermally amplified.
The flow
cell is a glass slide with lanes.
Each lane is a channel coated with a lawn,
composed of two types of oligos.
Hybridization is enabled by the first of the
two types of oligos on the surface.
This oligo
is complimentary to the adapter region on
one of the fragment strands.
A polymerase creates a complement of the
hybridized fragment.
The double stranded
molecule is denatured and the original
template is washed away.
The strands are
clonally amplified through bridge
amplification.
In this process the strand folds
over and the adapter region hybridizes to the
second type of oligo on the flow cell.
Polymerases generate the complimentary
strand forming a double stranded bridge.
This bridge is denatured, resulting in 2 single
stranded copies of the molecule that are
tethered to the flow cell.
The process is then repeated over and over,
and occurs simultaneously for millions of
clusters resulting in clonal amplification of all
the fragments.
After bridge amplification the
reverse strands are cleaved and washed off,
leaving only the forward strands.
The three
prime ends are blocked to prevent unwanted
priming.
Sequencing begins with the extension of the
first sequencing primer to produce the first
read.
With each cycle, fluorescently tagged
nucleotides compete for addition to the
growing chain.
Only one is incorporated
based on the sequence of the template.
After the addition of each nucleotide the
clusters are excited by a light source and a
characteristic fluorescent signal is emitted.
This proprietary process is called
Sequencing-by- Synthesis.
The number of
cycles determines the length of the read.
The emission wave length, along with the
signal intensity, determines the base call.
For a given cluster, all identical strands are
read simultaneously.
Hundreds of millions of clusters are
sequenced in a massively parallel process.
This image represents a small fraction of the
flow cell.
After the completion of the first read, the
read product is washed away.
In this step
the index 1 read primer is introduced and
hybridized to the template.
The read is
generated, similar to the first read.
After completion of the index read, the read
product is washed off, and the three prime
ends of the template are deprotected.
The
template now folds over and binds the
second oligo on the flow cell.
Index 2 is read in the same manner as index
1.
Polymerases extend the second flow cell
oligo forming a double stranded bridge.
This
double stranded DNA is then linearized and
the three prime ends are blocked.
The
original forward strand is cleaved off and
washed away leaving only the reverse
strand.
Read 2 begins with the introduction of the
read 2 sequencing primer.
As with read 1,
the sequencing steps are repeated until the
desired read length is achieved.
The read 2
product is then washed away.