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ULTRASEQUENCING . Next Generation Sequencing : methods and applications .

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

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ULTRASEQUENCING . Next Generation Sequencing : methods and applications .

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  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

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