NEW SEQUENCING TECHNOLOGY

S@NDEEP YADAV

Content• INTRODUCTION• NEW SEQUENCING TECHNOLOGY- A

COMPARISION • PYROSEQUENCING• Roche/454 Technology• ILLUMINA SOLEXA TECHNOLOGY• REFRENCE

SOLEXA

INTRODUCTION

• Developing new methods for fast polynucleotide sequencing• Quick identification of pathogens to save many lives• Sequencing has important medical applications• Sequencing methods proposed by Fredrick Sanger, Maxim & Gilbert• Dideoxy method was accepted because of easiness• Human genome project cost 3 billion US dollars and took 13 years to complete.

454 GS FLX

New Sequencing Technology

Roche 454 GS FLX

Illumina SOLEXA

Applied Biosystems SOLID

VisiGen Biotechnologies

Helicos BioSciences

Pyrosequencing

Sequencing by synthesis

Sequencing by ligation

Single-molecule sequencingNanopore sequencing

SEQUENCING TECHNOLOGY- A COMPARISION

Oligo ligation-cleavage

1000-1500 million

35 bases

2-4 days

$ 5,000 per run

Labeled base addition

1000-3000 million

35-50 bases

2-3 days

$ 3,500 per run

Roche/454 ABI Solid Illumina/Solexa

Sequencing method

Base per run

Read length

Length of run

Cost of just sequencing

Pyrosequencing chemistry

100 million

100-200 bases

7.5 hrs.

$ 9,000 per run

PYROSEQUENCING• Sequencing primer is hybridized to PCR amplified, ssDNA template

• Incubated with DNA pol, ATP sulfurylase, luciferase and apyrase, (substrate) adenosine 5´ phosphosulfate (APS) & luciferin.

• First four dNTP is added to the reaction

• DNA pol catalyzes the incorporation of complementary base in the template strand

• Each incorporation event will release pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide

• APS + PPi ATP + Sulphate •Luciferin + ATP oxyluciferin +

visible light

•Light produced is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™. Each light signal is proportional to the number of nucleotides incorporated.

PYROSEQUENCING

• Apyrase continuously degrades unincorporated dNTPs. Now another set of dNTPs added

• Note that deoxyadenosine alfa-thio triphosphate (dATPS) substitutes the natural deoxyadenosine triphosphate (dATP).

• As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the Pyrogram® trace.

1. Genome is loaded into a PicoTiter™ plate

2. Load PicoTiter plate into instrument

3. Load Reagentsin a single rack

4. Sequence EntireGenome at once,in real-time

454 Sequencing Instrument Ease of Use

454 LifeSciences Sequencer

GS FLX sequencer sequence the genomic DNA, PCR products, BACs and cDNA.

•Longer sequences are first sheared into random library of 300-800 bp long fragments.

•Adaptors added to both ends of the fragments. If sample is double stranded one strand is removed and the remaining single strands are used in the following steps

454 LifeSciences Sequencer

•Aided by the adaptors individual fragments are captured on their own unique beads. A bead and the bound fragment together with a water-in-oil emulsion form a microreactor so that each fragment can be amplified without contamination via the so called emulsion PCR (emPCR). The entire fragment collection is amplified in parallel via emPCR.

•Now the emulsion shell is broken and the clonally amplified beads are ready for loading onto the fibre-optic PicoTiterDevice for sequencing.

•Sequencing is accomplished by synthesizing the complementary strands of the bead attached templates. In a number of cycles the four bases (ATGC) are sequentially washed over the PicoTiterPlate. The incorporation of a new base is associated with the release of inorganic pyrophosphate starting a chemical cascade. This results in the generation of a light signal which is captured by a CCD camera.

•The PicoTiter Plate is loaded with one fragment carrying bead per well and smaller beads with the enzymes necessary for sequencing.

454 LifeSciences Sequencer

• Real Time Sequencing by Synthesis • Chemiluminescence detection in picotiler plates• Amplification: emulsion PCR• Pyrosequencing• up to 400,000 reads / run• on average 250 bases / read • up to 100 Mb / run• 99% accuracy at the 400th base• G-C rich content is not as much of a problem• capable of detecting mutations in an amplicon pool at a high sensitivity level, which may have implications in clinical research, especially cancer and HIV

454 LifeSciences Sequencer

ILLUMINA SOLEXA Flow Cell

ILLUMINA SOLEXA SEQUENCER

ILLUMINA SOLEXA SEQUENCER Contd.

Illumina / Solexa: Genetic Analyzer

• Real Time Sequencing by Synthesis

• Clonal Single Molecule Array

• Amplification: bridging PCR

• 60 mio reads / run

• up to 50 bases / read

• 2 Gb / run

• 8 channels, app. 5 mio reads / channel

• Fluorescent labels

• Reversible 3‘OH blocking

PRO

APPLICATION OF GEMONE ANALYSER

• de novo sequencing• cDNA libraries, ESTs• amplicon sequencing, long range PCR fragments• human samples• BAC pools• fosmid pools• metagenomes, biofilm• transcriptome

REFRENCE

• www.illumina.com• www.en.wikipedia.org/wiki/DNA_sequencing

• www.ncbi.nlm.nih.gov/ • www.454.com • www.genengnews.com/news

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