montefiore institute ulg - sequencing technologieskbessonov/archived_data/gbio009... ·...
TRANSCRIPT
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.