New Sequencing Technology

NEW SEQUENCING TECHNOLOGY

S@NDEEP YADAV

Content

SOLEXA

• INTRODUCTION • NEW SEQUENCING TECHNOLOGY- A COMPARISION • PYROSEQUENCING • Roche/454 Technology • ILLUMINA SOLEXA TECHNOLOGY • REFRENCE

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

Pyrosequencing

Illumina SOLEXA

Sequencing by synthesis

Applied Biosystems SOLID VisiGen Biotechnologies Helicos BioSciences

Sequencing by ligation Single-molecule sequencing Nanopore sequencing

SEQUENCING TECHNOLOGY- A COMPARISION Roche/454

ABI Solid

Illumina/Solexa

Sequencing method

Pyrosequencing chemistry

Oligo ligationcleavage

Labeled base addition

Base per run

100 million

1000-1500 million

1000-3000 million

Read length

100-200 bases

35 bases

35-50 bases

Length of run

7.5 hrs.

2-4 days

2-3 days

Cost of just sequencing

$ 9,000 per run

$ 5,000 per run

$ 3,500 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.

454 Sequencing Instrument Ease of Use 3. Load Reagents

2. Load PicoTiter plate into instrument

in a single rack

4. Sequence Entire Genome at once, in real-time

1. Genome is loaded into a PicoTiter™ plate

454 LifeSciences Sequencer

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

•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.

454 LifeSciences Sequencer

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

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

ILLUMINA SOLEXA

Flow Cell

ILLUMINA SOLEXA SEQUENCER

ILLUMINA SOLEXA SEQUENCER Contd.

Illumina / Solexa: Genetic Analyzer PRO • 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

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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