2. NGS
Also known as high-throughput DNA
sequencing tehniques.
Developed in 2005.
Perform numerous sequencing reactions
simultaneously.
Less expensive.
Quick.
3. 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)
5. 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.
7. 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.
8. 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.
10. 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.
12. 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.
13. ILLUMINA / SOLEXA GENOME ANALYZER
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.
15. ILLUMINA / SOLEXA GENOME ANALYZER
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.
18. APPLIED BIOSYSTEMS SOLID
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.
19. APPLIED BIOSYSTEMS SOLID
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.