Download Illumina Solexa

Illumina Solexa
Solexa Genome Analyzer IIe
> 20 Gb of paired-end data per run
HiSeq 2000
~30x coverage of two human
genomes in a single run
Illumina Solexa
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Solid state amplification
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Use of reversible terminator nucleotides
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DNA fragments are bound to a solid surface
Bridge amplification occurs on the slide
Fluorescent dye on the 30 OH end of each nucleotide.
Output:
Original
technology
Reads
Run time
Output
35bp
90hrs
1 ×109
HiSeq 2000
1 × 36bp
48hrs
105 ×109
2 × 50bp
132hrs
300 ×109
2 × 100bp
264hrs
600 ×109
Prepare genomic DNA
Randomly fragment
genomic DNA and ligate
adapters to both ends of
the fragments.
Attach DNA to surface
Bind single-stranded
fragments randomly to
the inside surface of the
flow cell channels (8).
Bridge amplification
Add unlabelled
nucleotides and enzyme
to initiate solid-phase
bridge amplification.
Make double-stranded
The enzyme incorporates
nucleotides to build
double stranded bridges.
Denature the DNA
Denaturation leaves
single stranded templates
anchored to the
substrate.
Complete amplification
Several million dense
clusters of double
stranded DNA are
generated within each
channel of the flow cell.
Determine first base
Add all four labeled
reversible terminators,
primers and polymerase
to the flow cell.
Image first base
After laser excitation,
capture the image of
emitted fluorescence from
each cluster on the flow
cell.
Determine second base
Add all four labeled
reversible terminators,
primers and polymerase
to the flow cell.
Image second base
After laser excitation,
capture the image of
emitted fluorescence from
each cluster on the flow
cell.
Cycle
Repeat over again,
capturing a single base at
a time.
Analyze data
Align data, compare to
reference sequence and
identify sequence
differences.
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Genomic DNA
shear to 200−500bp fragments
Ligate adapters
A1 SP1
SP2
A2
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Generate clusters
SP2
A2
SP1 A1
Flow cell
Genomic DNA
shear to 200−500bp fragments
Ligate adapters
A1 SP1
Sequence
A2
SP1
SP2
A2
A1
Flow cell
SP2
Genomic DNA
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shear to 2−5kb fragments
Biotinylate 5’ ends
Biotin
Biotin
Circularize
Fragment 400−600bp
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Genomic DNA
Affinity purify biotin labels
shear to 2−5kb fragments
Biotinylate 5’ ends
Ligate adapters
Biotin
A1 SP1
Biotin
SP2
Circularize
A2
Generate clusters
A2
SP1 A1
Flow cell
SP2
Sequence
SP1
SP2
A2
A1
Flow cell
Fragment 400−600bp
You can get a review of this at
www.youtube.com/embed/HMyCqWhwB8E?iframe&rel=
0&autoplay=1
or many other videos ...
Summary older systems - Sanger
Specifications:
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Read length: 0.6 to 1.5 kb
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Throughput: 1X (one sequence/sample)
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Direction: Forward or reverse
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Cost per sample: 4 to 5$ per sample
Limitations:
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No multiplexing
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Some problems with secondary structure
Applications:
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Low overall run cost
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Single gene/short fragment sequencing
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Clone/marker sequence
Summary older systems - Illumina GAII
Specifications:
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Read length: 30-75 bp
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Throughput: 20Gb per run
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Direction: Forward or reverse
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Cost per sample: 10K$ per run
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Number of lanes per cell: 8 lanes
Limitations:
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Short read length
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Longer runs (1-2 weeks)
Applications:
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High throughput for shorter fragments
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Gene expression studies
Ion Torrent sequencing - semiconductor technology
When a nucleotide is incorporated into a strand of DNA by a polymerase, a
hydrogen ion is released as a byproduct.
From www.iontorrent.com
Ion Torrent sequencing - semiconductor technology
A high-density array of wells, each holding a different DNA template. Beneath
the wells is an ion-sensitive layer and then a proprietary Ion sensor.
From www.iontorrent.com
Ion Torrent sequencing - semiconductor technology
When a C nucleotide is incorporated a hydrogen ion will be released. The
charge from that ion can be detected by the sensor and changed to digital
information.
Ion Torrent sequencing - semiconductor technology
Nucleotides are sequentially added. If the next nucleotide is not a match, no
voltage change will be recorded and no base will be called.
From www.iontorrent.com
Ion Torrent sequencing - semiconductor technology
With two identical bases, the voltage will double. There is no scanning, no
cameras, no light - each nucleotide incorporation is recorded in seconds.
From www.iontorrent.com
Pacific Biosci sequencing
In this method, a single polymerase is followed as it sequences just
one template. There is no amplification. The four nucleotides with
four separate fluorophores are all present and followed over time.
Since it is the native polymerase (albeit modified), very long reads
can be obtained but since it is a single molecule, error rates are
high.