Conventional and next generation sequencing ppt

CONVENTIONAL AND NEXT

GENERATION SEQUENCING

Submitted by:

Ashwini.R, Kaveri Singh and Preeti Sharma-

Amity Institute of Biotechnology

WHAT IS GENE SEQUENCING?

DNA sequencing is the process of determining the sequence of

nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA.

Sequencing an entire genome (all of an organism’s DNA)

remains a complex task. It requires breaking the DNA of the

genome into many smaller pieces, sequencing the pieces, and

assembling the sequences into a single long "consensus."

However, thanks to new methods that have been developed over

the past two decades, genome sequencing is now much faster

and less expensive.

METHODS OF SEQUENCING

Classical sequencing

Maxam Gilbert – Chemical Breakdown Method

Sanger Coulson – Dideoxy or chain termination

method

Next generation sequencing

Pyrosequencing

Virtual terminator sequencing

SoLid

MAXAM GILBERT OR CHEMICAL BREAKDOWN

METHOD

1. DNA to be sequenced is end labeled

usually at the 5’ end.

2. Dephosphorylation by alkaline

phosphatase – removes 5’ phosphate

3. Phosphorylation using labeled ATP and

kinase.

4. Removal of label from one of the 5’ ends.

The labeled template is chemically

broken by two sets of reactions – one

specific for purines, and another for

pyrimidines

The labeled template is chemically broken by two sets of

reactions – one specific for purines, and another for

pyrimidines

SANGER SEQUENCING: THE CHAIN TERMINATION

METHOD

Sanger sequencing is a DNA sequencing method in which target

DNA is denatured and annealed to an oligonucleotide primer,

which is then extended by DNA polymerase using a mixture of

deoxynucleotide triphosphates (normal dNTPs) and chain-

terminating dideoxynucleotide triphosphates (ddNTPs).

ddNTPs lack the 3’ OH group to which the next dNTP of the

growing DNA chain is added. Without the 3’ OH, no more

nucleotides can be added, and DNA polymerase falls off. The

resulting newly synthesized DNA chains will be a mixture of

lengths, depending on how long the chain was when a ddNTP

was randomly incorporated.

AUTOMATED SANGER SEQUENCING

Use of differentially labeled fluorescent

ddNTPs facilitates resolving all fragments in a

single lane instead of four for each sample.

Differential labeling also increases the read

length as the bands can be allowed to flow out

of gel post detection.

Uses Capillary electrophoresis - higher

resolution, faster run.

Higher parallelism - 96 samples in one run.

Sequence output - chromatogram

NEXT-GENERATION

SEQUENCING

Also known as high-throughput sequencing, is the term used to describe a number of different modern sequencing technologies including:

Roche 454/ Pyrosequencing sequencing

Virtual Terminator sequencing

SOLiD sequencing

These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such have revolutionised the study of genomics and molecular biology.

APPLICATIONS OF NEXT-GENERATION SEQUENCING

Full-genome resequencing or more targeted

discovery of mutations or polymorphisms.

Large-scale analysis of DNA methylation.

Clinical genetics: There are numerous opportunities

to use NGS in clinical practice to improve patient

care, including:

1. NGS captures a broader spectrum of mutations

than Sanger sequencing.

2. Genomes can be interrogated without bias.

SOLID (SUPPORT OLIGO LIGATION AND

DETECTION)

Ligase based sequencing.

Instead of dNTPs we add (8bp) oligo nucleotides.

Uses an adapter-ligated fragment library.

Uses an emulsion PCR approach with small

magnetic beads to amplify the fragments for

sequencing.

Read lengths for SOLiD are user defined between

25–35 bp, and each sequencing run yields between

2–4 Gb of DNA sequence data.

STEPS INVOLVED

Basically it involves 7 steps:

1. Attaching of the primer

2. Ligation

3. Imaging

4. Capping unextended strands

5. Cleaving off fluorophores

6. Repeating steps 2-5 to extend the sequence

7. Melting off extended sequence

DETAILED PROCESS

1.Sequencing Library Preparation and Immobilization

Fragmentation of the DNA sample to an appropriate size range (400–850 bp), end repair and ligation of “P1” and “P2” DNA adapters to the ends of the library fragments.

Emulsion PCR is applied to immobilize the sequencing library DNA onto “P1” coated paramagnetic beads.

Immobilization of the modified beads to a glass slide is done.

The glass slides can be segmented up to eight chambers to facilitate up scaling of the number of analyzed samples.

Preparation of Emulsion PCR

Execution of Emulsion PCR

STEPS INVOLVED

2.Sequencing by Ligation

A sequencing primer is hybridized to the “P1”

adapter in the immobilized beads.

A pool of uniquely labeled oligonucleotides contains

all possible variations of the complementary bases

for the template sequence.

It applies partially degenerate, fluorescently

labeled, DNA octamers with dinucleotide

complement sequence recognition core.

These detection oligonucleotides are hybridized to

the template and perfectly annealing sequences are

ligated to the primer.

Dinucleotide octamers

STEPS INVOLVED

After imaging, unextended strands are capped and

fluorophores are cleaved.

A new cycle begins 5 bases upstream from the

priming site.

After the seven sequencing cycles first sequencing

primer is peeled off and second primer, starting at

n-1 site, is hybridized to the template. In all, 5

sequencing primers (n, n-1, n-2, n-3, and n-4) are

utilized for the sequencing.

As a result, the 35-base insert is sequenced twice

to improve the sequencing accuracy.

PYROSEQUENCING

It is a type of Modern generation sequencing

Read length around 400 bp

Very fast sequencing process

Uses an enzymatic system

STEPS OF PYROSEQUENCING

The genomic DNA is first cut into the fragments of

300-800 bp

Small adapters are added to each ends of the

fragments

Double stranded DNA denatured to a single stranded

DNA

MICROBEADS

Microbeads are coated with DNA Primers (microbeats are

20 microns in diameter)

Complementary to one of the adapters

Single stranded DNA molecules attaches to the primer

(By complementary base pairing)

Bead are present in the Emulsion =oil + Reaction

Mixture

(DNA)n + dNTP polymerase (DNA) n+1 + Ppi

pyrophosphate is released

Apyrase – Degrade the extra nucleotides before

the next cycle begins

ADVANTAGES

Massive parallel sequencing

Less time taking (One genome sequencing done in

7 days)

cheaper

Virtual terminator sequencing

The virtual terminator (VT)

nucleotides are nucleotide analogues,

contain a fluorescent dye and

chemically cleavable group (VT) – 3’-

O-azidomethyl group.

Once incorporated, the VT analogues

block further incorporation until the

VT moiety is chemically removed.

The virtual terminator label is

removed before the next cycle ,

regenerating the 3’ OH group using

reducing agent tris 2 carboxy ethyl

phosphine (TCEP)

Primer is extended through virtual terminator nucleotides by adding all

four differentially labelled nucleotides simultaneously

Because of the VT moitey only one nucleotide is added at a time and

recorded for each sequence based on differential florescence tagging.

Next the virtual terminator moitey is removed, regenerating the 3’ OH

group using reducing agent tris 2 carboxy ethyl phosphine (TCEP)

These steps are repeated till the template is fully sequenced

THANK YOU