next generation sequemcing

Next generation sequencing
By Shahzeb khan

NGS
Also known as high-throughput DNA
sequencing tehniques.
Developed in 2005.
Perform numerous sequencing reactions
simultaneously.
Less expensive.
Quick.

NGS
 widely used sequencers;
1. The bead-amplification sequencing
(Roche/454FLX).
2. Sequencing by synthesis (Illumina / Solexa
Genome analyzer).
3. Sequencing by ligation (Applied Biosystems
SOLID System)

NGS
Roche 454
Ilumina/
solexa GA
SOLID

NGS
All NGS systems have three common features differing
on the techniques used;
1. Sample preparation(fragmentation/ligation of
adapters).
2. Amplification(bridge PCR/emulsion PCR).
3. Analysing results.

UNIVERSAL LIBRARY PREPARATION.

ROCHE/454FLX
First next-generation sequencing technology.
By 454 Life Science (Branford, CT, USA) in 2005.
Read length 700 Bp.
Use pyrosequencing technique.
Sequencing by synthesis principle.

ROCHE/454FLX
EMULSION PCR
Adapters, ligated at both end of DNA fragments.
Fragments are mixed with small 28-µm streptavidin-
coated beads.
Beads have sequences complementary to adapters.
For fragment ampliﬁcation all required reagents are
provided in droplets of water in oil mixture .
Each bead now carry millions of sequences.
Each bead must carry a unique sequence.

ROCHE/454FLX
SEQUENCING
Beads incubated with DNA-polymerase are loaded onto
PTP well.
PTP wells are filled with 1µm bead having sulfurylase
and leuciferase.
PTP wells are loaded onto 454FLX sequencer provided
with dNTPS and buffers.
Four nucleotides are added in sequential manner.
When a nucleotide is incorporated a light signal is picked
by CCD camera.

ILLUMINA / SOLEXA GENOME ANALYZER
introduced by Solexa in 2006 (San Diego, CA, USA)
read length to up to 100 Bp.
Use bridge PCR technique.
Sequencing by synthesis principle.

BRIDGE PCR
Adapters, ligated at both end of DNA fragments.
Fragments, immobilised on a flow cell having
primers complementary to adapters.
A bridge structure is created.
After several PCR cycles 1000s of s.s DNA
fragments are synthesised.

SEQUENCING
We need homogenous population of strands for
sequencing.
The cluster of fragments are supplied onto another
surface having primers.
Four reversible blocked nucleotides (3´-OH is chemically
blocked) are added.
After the acquisition of images in each cycle, the 3´-OH
blocking group is chemically removed.
 Another cycle can be initiated.

APPLIED BIOSYSTEMS SOLID
Developed in late 2007.
Use pyrosequencing technique for amplification.
Sequencing by ligation principle.

Amplification of fragments through EMULSION PCR as
discussed earlier.
SEQUENCING
Beads having unique DNA fragments are fixed on a flow
cell via 3´- modiﬁcation of DNA strands.
Primers are annealed complementary to adapters.
Fluorescently labelled octamers are added to the
mixture.
Each octamer also holds one of four ﬂuorescent dyes.

Octamer whose speciﬁc dinucleotide(usually 4,5
nucleotide) is matched with a template is hybridized and
than ligated by ligase.
An image is taken at this stage.
 Last three bases(6,7,8) of octamer are chemically
removed, so ﬁve bases of octamer are left behind.
The above step is repeated 10 times.
The same cycle is repeated with another primer(n-1) for
bases (3,4) and so on.

next generation sequemcing

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next generation sequemcing