Sequencing TechnologiesThe next generation

● Old Sander technology for 20 years

● NGS brings a quicker way to do sequencing

● With NGS, in the future you could have your personal genome sequencing, in an inexpensive way.

Next-Generation Sequencing

● Template preparation

● Sequencing and Imaging

● Genome alignment and assembly

● Produces large and inexpensive data

NGS, continued

● Roche/454, Illumina/Solexa, Life/APG, Helicos BioSciences, Polonator and the near-term Pacific Biosciences

● Each has its specific protocol○ makes it difficult to compare○ or to do quality analyses

NGS, different platforms

● Two methods

○ clonally amplified templates■ emPCR■ solid-phase

○ single-molecule templates■ Immobilize the template■ Immobilize the primer■ Immobilize the polymerase

NGS, Template preparation

emPCR

Solid Phase

Template immobilized

Primer immobilized

Polymerase immobilize

● Chosen method matters a lot● Errors occur depending on the template

preparation method and sequencing method

● Choice depends on application

NGS, sequencing and imaging

● Four strategies :○ Cyclic reversible termination (CRT)○ Sequencing by ligation (SBL)○ Single-nucleotide addition (pyrosequencing) ○ Real-time sequencing

● Sequence alignment○ compare to reference sequence or ○ assemble de novo

NGS, sequencing and imaging

CRT (4-colour)Illumina/Solexa

Sequencing byligationLife/APG

PyrosequencingRoche/454

Real time sequencingPacific Biosciences

● Illumina/Solexa and Life/APG : Re-sequencing human genome

● Helico BioSciences : RNA-seq

NGS, suitable applications

● Aim to catalogue○ Single-nucleotide variants (SNV)○ Structural variants (SV)○ How they are associated to the phenotype

● Personal genomics for medical purpose

● We still have to make it affordable while maintaining the high-quality standard

Personal genomes

● Still expensive● Should be done for particular regions of

the genome● PCR is only for small scales

○ microdroplet PCR technology ○ oligonucleotide microarrays○ solution-based hybridization

Personal sequencing

Some numbers

Platform Number of reads

Genome coverage

Number of runs

Cost

Sanger 31.9 millions N/A >340 000 70 000 000 $

Roche/454 93.2 millions 95 % 234 1 000 000 $

Illumina/Solexa 1 647 millions 99.9 % 35 500 000 $

Helicos BioSciences 2 725 millions 90 % 4 48 000 $

● What will we do with this enormous lot of data ?○ How to store it ?○ How to transfer it ?

● Personal genomics○ Will it be affordable ?○ Will it answer people's questions ?○ It's already happening○ What about genome privacy ?

There are still some questions

Maybe we can answer some questions !

And thank you for your attention.