GENOMICA STRUTTURALE: GENOMICA FUNZIONALE: 1. Anatomia dei genomi. 8. Funzionamento dei genomi Il genoma dei procarioti

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1 GENOMICA STRUTTURALE: GENOMICA FUNZIONALE: 1. Anatomia dei genomi 8. Funzionamento dei genomi Il genoma dei procarioti Modificazioni della cromatina e l espressione del genoma Il genoma degli eucarioti Microarray e RNA-seq 2. La mappatura dei genomi Metil-seq Mappatura genetica Chip-seq Mappatura fisica 3. Il sequenziamento automatico del DNA Il principio del sequenziamento secondo Sanger Il sequenziamento su larga scala La lettura dei tracciati di sequenziamento 4. Il sequenziamento del genoma I nuovi metodi di sequenziamento Il sequenziamento gerarchico Il sequenziamento shogun Piattaforme di sequenziamento di interi genomi La verifica delle sequenze e assemblaggio dei contig 5. L annotazione del genoma Il sequenziamento delle EST Le caratteristiche funzionali delle sequenze genomiche 6. I Progetti Genoma Il Progetto Genoma Umano Il Progetto Genoma Animali Il Progetto Genoma Piante Il Progetto Genoma Microrganismi 7. Genotipizzazione Gli SNP e la variazione La genotipizzazione degli SNP

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3 Sequenziamento per shotgun gerarchici Sequenziamento per shotgun dell intero genoma

4 High-throughputSequencing 1. First Generation Sequencing 2. Second (Next) Generation Sequencing 3. Third Generation Sequencing Cyclic-array sequencing based

5 First Generation Sequencing Sanger sequencing based For shotgun sequencing DNA is fragmented Clonedintoa plasmidvector Cyclic sequencing reaction Separation by electrophoresis Readout with fluorescent tags many clonal copies of a single plasmid insert within a spatially isolated bacterial colony

6 Next Generation Sequencingg Cyclic-array sequencing method For shotgun sequencing DNA is fragmented Adaptors ligated to fragments ArrayofPCR colonies Enzymatic extension with fluorescently tagged nucleotides Cyclic readout by imaging array manypcr ampliconspresentwithinsingle reaction volume

7 Polimerase colony Èuna amplificazione clonale, in vitro, di una singola molecola di DNA

8 Polony Polonies are colonies of PCR amplicons derived from a single molecule of nucleic acid. Thousands to millions of spatially distinct polonies, each with an effective reaction size on the order of nanoliters (10-9 L) to femtoliters (10-15 L), can be amplified simultaneously within an acrylamide gel. Enzymatic reactions on polonies (e.g. in situ hybridization; minisequencing) can be subsequently performed in parallel. PCR in emulsione PCR tramite ponti

9 Emulsion PCR Fragments, with adaptors, are PCR amplified within a water drop in oil. One primer is attached to the surface of a bead. Used by 454, SOLiDand Polonator.

10 Bridge PCR DNA fragments are flanked with adaptors. A flat surface coated with two types of primers, corresponding to the adaptors. Amplification proceeds in cycles, with one end of each bridge tethered to the surface. Used by Solexa(Illumina).

11 454 sequencing based on pyrosequencing chemistry (sequencing by synthesis),, uses an emulsion-based method to isolate and amplify DNA fragments in vitro. Fragments of nt in length are ligated to special DNA Capture Beads, one fragment per bead. The beads are captured in water droplets of a heat-stable water-in-oil emulsion; one bead per droplet. Amplification occurs in the droplets, containing PCR-reagents (microreactors). The 454 GS FLX platform gives read lengths of ~250bp, providing about 400,000 sequences and Mb per run.

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19 Illumina Solexa sequencingtechnology is a platform based on massively parallel sequencing of millions of fragments using reversible terminator-based sequencing chemistry (modified Sanger). This technology relies on fragmented genomic DNA arranged on a planar, optically transparent surface. Attached DNA fragments are amplified to create an ultra-high density sequencing flow cell with Illumina Sole xa seq uenc ing techno logy is a platform based on massively parall el seq uenc ing of millions of fragmen ts using reversible terminatorbased sequenc ing of chem istry the (mod ified same Sanger). This techno template logy relies on fragmen(up ted genom ic to DN A arra ten nged on a million planar, op tically transpsingle-molecule arent surface. ~1,000 copies Attached DN A fragmen ts amplified to create an ultra -high dens ity sequencing flow cell with ~1,000 cop ies of the same temp late (up to ten million clusters per single-molecule clusters per square These temp lates are seq uenced using a four-color DN A seq uenc ing-by-syn thes is technology that emp loys square reve rsible terminators centimetre). with remov able fluoresc dyes. The These Solexa platform provides templates up to 70bp pe r read, with are a paired sequenced end capability of using a ~3050 million reads and 24G b per run. four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. The Solexa platform provides up to bp per read, with a paired end capability of ~3050 million reads and 24Gb per run.

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24 Illumina Flow Cell One flow cell Eight 1.4-mm wide channels Each channel can run up to twelve differently tagged libraries (Multiplexed Sequencing) Input requirement: μg (single- and paired-end reads), 10 μg(mate Pair reads) million reads (clusters) per flow cell, each containing ~1,000 copies of the same template

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26 ABI SOLiD System is a highly accurate, massively parallel genomic analysis platform. Based on sequencing by ligation, generates DNA by measuring the serial ligation of an oligonucleotide. The SOLiD System generates over 20 gigabases and 400M tags per run, with system accuracy greater than 99.94%, due to 2 base encoding which enables unique error checking capability, providing higher confidence ineach call.

27 Library preparation

28 Emulsion PCR/Bead Enrichment

29 Bead Deposition

30 Sequencing by Ligation/Data Analysis

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33 Sequenze corte Sequenzecorte, ma tecnologiain continua evoluzione: 454: 100 basi ? Solid: 25 basi ? Illumina: 32 basi ? Difficoltà di assemblare sequenze corte de novo, soprattutto per il problema delle sequenze ripetute complicato ancora di più rispettoa Sanger (lunghezzamedia 600bp)

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35 The Polonator

36 SequenziamentoSangerad alta processività PREPARAZIONE DELLA LIBRERIA Frammentazione casuale del DNA genomico clonazione e trasformazione in batteri 7-10 giorni assumendo di possedere una piattaforma robotica per alta processività Raccolta delle colonie Purificazione del DNA dalle colonie Sequenziamento Sanger Elettroforesi capillare Settimane-anni, dipendentemente dalle dimensione del genoma (e copertura richiesta) e dal numero di sequenziatori capillari a disposizione Mappatura delle reads su un genoma di riferimento (o assemblaggio de novo)

37 Sequenziamentodi Nuova Generazione PREPARAZIONE DELLA LIBRERIA Frammentazione casuale del DNA genomico Ligazione degli adattatori 1-3 giorni Amplificazione clonale dei frammenti Sequenziamento nuova generazione Processamento delle immagini 1-7 giorni Mappatura delle reads su un genoma di riferimento (o assemblaggio de novo)

38 High-throughput Sequencing 1. First Generation Sequencing 2. Second (Next) Generation Sequencing 3. Third Generation Sequencing

39 Nanopore sequencing Nucleic acids driven through a nanopore Differences in conductance of pore provide readout Real-time monitoring of PCR activity Read-out by fluorescence resonance energy transfer between polymerase and nucleotides (VisiGen Biotechnologies approach) Waveguides allow direct observation of polymerase and fluorescently labeled nucleotides (Single Molecule SMRT sequencing)

40 Ion semiconductor sequencing DNA nanoball sequencing Helioscope(TM) single molecule sequencing Single Molecule SMRT(TM) sequencing Single Molecule real time(rnap) sequencing Nanopore DNA sequencing VisiGen Biotechnologies approach

41 cyclodextrin α-hemolysin Nanopore sequencing

42 IonTorrentSequencing

43 HeliScopeSingle MoleculeSequencertechnology True Single Molecule Sequencing (tsms) is a powerful new method capable of directly measuring single DNA molecules, without amplification. Helioscope sequencing uses DNA fragments with added polya tail adapters, which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The incorporated nucleotides emit light that is detected by the HeliScope Single Molecule Sequencer. Tracking nucleotide incorporation on each strand determines the exact sequence of each individual DNA molecule. The reads are short, up to 55 bases per run