1 / 10

ULTRASEQUENCING . Next Generation Sequencing : methods and applications .

ULTRASEQUENCING . Next Generation Sequencing : methods and applications . Genòmica i Proteòmica Pablo Lammers

teleri
Download Presentation

ULTRASEQUENCING . Next Generation Sequencing : methods and applications .

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. ULTRASEQUENCING.NextGenerationSequencing: methods and applications. GenòmicaiProteòmica Pablo Lammers Curs 12/13 NIU 1323099

  2. Sanger sequencing • Since1975. Frederick Sanger • Human GenomeProject • New necessities • 1 sample -> 1 read -> 3 to 9€ • 1 read -> 1 kbp (max.) • 1 run -> 16/48/96samples

  3. NextGenerationSequencing DNA Librariespreparation Sonication Physicalmethods Fragmentation Amplification Chemicalmethods Adaptersligation Sequencingreaction • 1 run -> 1000 € • 1 read -> 100 to 400 bp • 1 run -> >100 M reads • 1 run -> 24 smallgenomes

  4. NGS platforms 454 Roche – GS Junior AppliedBiosystems: SOLiD Illumina- MiSeq Invitrogen – Ion Torrent PacificBiosciences – PacBio RS II

  5. 454 (Roche) • Onefragment = Onebead • emPCR: Emulsion PCR amplification • Sequencing: Onebead = Oneread • Pyrosequencing • 1 M reads/run • Readlenght: 250-500 bp DNA capturedbeadcontainingmillions of copies of a single clonallyamplifiedfragment Library construction emPCR PTP loading

  6. AppliedBiosystems: SOLiD • AmplificationbyemPCR • Hybridizationtobeads. Beadscovalentlyattachedtoglassslide. • LigationBasedSequencingwithDi-Base probes(fluorescentlylabeledwith 4 dyes) • Image capture (fluorophore) • 100-500 M reads/run • Readlenght: 50-100 bp

  7. Illumina Sequencingbysynthesis 3. Sequencing 2. Clusters generation 1. Library preparation DNA fragmentation Adapteroligosligated Isothermal bridge amplification Purification • 100 M reads/run • Readlenght: 80-250 bp

  8. Ion Torrent wells -> chemicalinfofrom DNA seq -> into digital info (basecalls) DNA fragmented Attachedtobeads Eachbead in a well one of the 4 nucleotides Nucleotideincorporatedto a single DNA strain ion H released pH chemichalchanges -> intovoltage • each 15 sec -> wash and repeat (differentnucleotide) • 10 M – 1 G reads/run • Readlenght: 200-400 bp

  9. PacificBiosystems (PACBIO) • Amplificationnotrequired • SMRT: Single Molecule Real Time seq • ZMW: Zero-modewaveguide DNA template-polymerasecomplex -> immobilized at thebottom of the ZMW Eachnucleotidelabeledwith a differentcoloredfluorosphore Phospholinkednucleotides -> introducedintothe ZMW chamber Base held -> light pulse produced • Readlenght: 4 kbp • Maximum: 23 kbp

  10. Applications • Cancerresearch • Populationgenomicsstudies • Metagenomics • RNA-seq • Comparativegenomics • Diseaseassociationstudies • Speciesclasification • Forensics

More Related