montefiore institute ulg - sequencing technologieskbessonov/archived_data/gbio009... ·...
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Sequencing TechnologiesThe next generation
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● 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
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● Template preparation
● Sequencing and Imaging
● Genome alignment and assembly
● Produces large and inexpensive data
NGS, continued
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● 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
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● Two methods
○ clonally amplified templates■ emPCR■ solid-phase
○ single-molecule templates■ Immobilize the template■ Immobilize the primer■ Immobilize the polymerase
NGS, Template preparation
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emPCR
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Solid Phase
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Template immobilized
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Primer immobilized
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Polymerase immobilize
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● Chosen method matters a lot● Errors occur depending on the template
preparation method and sequencing method
● Choice depends on application
NGS, sequencing and imaging
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● 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
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CRT (4-colour)Illumina/Solexa
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Sequencing byligationLife/APG
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PyrosequencingRoche/454
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Real time sequencingPacific Biosciences
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● Illumina/Solexa and Life/APG : Re-sequencing human genome
● Helico BioSciences : RNA-seq
NGS, suitable applications
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● 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
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● 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
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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 $
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● 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
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Maybe we can answer some questions !
And thank you for your attention.