lezione 5 - unifem.docente.unife.it/silvia.fuselli/dispense-corsi/... · lezione 5 next generation...
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Lezione 5
Next Generation Sequencing
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Perchè Next Generation Sequencing Si possono generare centinaia di milioni di corte sequenze (35bp-250bp) in una sola corsa in un tempo breve con un basso prezzo per base sequenziata. • Illumina HiSeq 2500, MiSeq, Next seq 500 • Life Technologies Ion Proton/Ion PGM • Applied Biosystems SOLiD e Roche/454 FLX, Titanium
Reviews: Michael Metzker (2010) Nature Reviews Genetics 11:31
Quail et al (2012) BMC Genomics Jul 24;13:341.
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Perchè bioinformatica
(wall.hms.harvard.edu)
Informatics
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Bioinformatica: sfide in NGS Data Analysis
• File di testo MOLTO GRANDI (migliaia di milioni di righe) – Non si possono usare gli strumenti “soliti”
– Enorme utilizzo di memoria e tempi di corsa
– Gestire, analizzare, accumulare, trasferire ed archiviare file giganteschi
• Necessità di computer potenti e di competenze – Computer clusters
– Necessità di nuovi algoritmi e software spesso open source Unix/Linux based.
– Collaborazione tra chi sviluppa la tecnologia, i bioinformatici e i biologi
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Basic NGS Workflow
Olson et al.
Library Samples preparation
Application specific
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Terminology • Coverage (depth): The number of nucleotides from reads that are
mapped to a given position. • Quality Score: Each called base comes with a quality score which
measures the probability of base call error.
• Paired-End Sequencing: Both end of the DNA fragment is sequenced, allowing highly precise alignment.
• Multiplex Sequencing: "barcode" sequences are added to each sample so they can be distinguished in order to sequence large number of samples on one lane.
• Mapping: Align reads to reference to identify their origin.
• Assembly: Merging of fragments of DNA in order to reconstruct the original sequence.
• Duplicate reads: Reads that are identical.
• Multi-reads: Reads that can be mapped to multiple locations equally well.
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Applications
Epigenetica
Espressione genica
Caratterizzazione regioni di interazione DNA-proteine
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Applications: genomes, exomes, transcriptomes
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Applications: RNA seq (trascrittoma)
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Applications:ChIP-Seq
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• Frammentazione
• Size selection
• Legame adattatori library
• Serve a reggiungere una quantità di DNA stampo sufficiente per la lettura del sequenziamento
Template preparation
• Sequencing by synthesis
• Lettura del segnale sequencing
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Preparazione del DNA o dell’RNA in una forma utilizzabile dalla tecnologia di
sequenziamento (lunghezza adeguata tipicamente poche centinaia di bp, dsDNA,
adattatori)
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Library preparation
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With the exception of Illumina’s Nextera prep, library preparation generally entails: (i) Fragmentation, size selection, end-repair, phosphorylation of
the 5´ prime ends, A-tailing of the 3´ ends to facilitate ligation to sequencing adapters
(ii) ligation of adapters (iii)some number of PCR cycles to enrich for product that has
adapters ligated to both ends. The primary differences in an Ion Torrent workflow are the use of blunt-end ligation to different adapter sequences.
(iv)To facilitate multiplexing, different barcoded adapters (index) can be used with each sample.
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Sonicazione: l'utilizzo di onde acustiche, in particolare ultrasoniche, condotta con l'ausilio di un sonificatore, un apparecchio che genera vibrazioni meccaniche amplificate sfruttando corrente elettrica ad elevata frequenza prodotta da un generatore. Gli ultrasuoni vengono trasmessi in una vasca contenente acqua.
The Covaris process is conducted under isothermal conditions, ensuring the integrity of the nucleic acid sample is maintained and providing high recovery of double-stranded DNA. Combined with the specifically engineered AFA Tubes it is possible to precisely and accurately fragment DNA and RNA to the 100 – 1500bp range (microTUBE), or 2 – 5kb range (miniTUBE).
DNA fragmentation: ultra sonication (COVARIS)
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Size selection: gel o beads magnetiche
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Illumina adaptors (gli adattatori servono a legare il frammento alla cella dove avviene l’amplificazione e il sequenziamento (P5/P7) e fungono da primer per la reazione di amplificazione prima e di sequenziamento poi) (Rd1 e Rd2 servono per il paired-end sequencing = sequenziamento a partire da entrambe le estremità di un frammento)
P5/ P7: binding sites to the flow cell Rd 1 SP: read1 sequencing primer Rd 2 SP: read2 sequencing primer
http://nextgen.mgh.harvard.edu/IlluminaChemistry.html
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Tagmentation (nextera): non rottura meccanica+ligazione adattatori, ma ‘tagmentazione’ enzimatica + inserzione adattatori
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Mate-pair sequencing • Pensato per affrontare il problema dei de novo
assembly e delle regioni complesse non leggibili con frammenti corti tipici dell’NGS
• Il paired-end seq costituisce già un passo avanti
• Punti chiave mate-pair: frammenti lunghi (2-10Kb) + estremità marcate con biotina + circolarizzazione + taglio random + recupero frammenti con biotina + sequenziamento delle due estremità
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Preparation of Illumina mate-pair libraries. Long fragments are end-repaired using biotinylated nucleotides (1). After circularization, the two fragment ends (green and red) become located adjacent to each other (2). The circularized DNA is fragmented, and biotinylated fragments are purified by affinity capture. Sequencing adapters (A1 and A2) are ligated to the ends of the captured fragments (3) and the fragments are hybridized to a flow cell, in which they are bridge amplified. The first sequence read is obtained with adapter A2 bound to the flow cell (4). The complementary strand is synthesized and linearized with adapter A1 bound to the flow cell, and the second sequence read is obtained (5). The two sequence reads (arrows) will be directed outwards from the original fragment (6).
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Library quality control and quantitation
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Prima del sequenziamento dobbiamo sapere se i frammenti hanno un certo range di lunghezze, se il DNA è di buona qualità e la concentrazione
• Fluorometer (Qubit)
• qPCR (real time PCR usando primer che riconoscono gli adattatori all’estremità dei frammenti della library)
• Agilent Bioanalyser
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Amplificazione clonale della library
(già nello strumento per NGS!)
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Metodologie principali
• Emulsion PCR (Roche 454, Ion torrent)
https://www.youtube.com/watch?v=bFNjxKHP8Jc
https://www.youtube.com/watch?v=MxkYa9XCvBQ
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Metodologie principali
• Solid-phase cluster generation (Illumina)
https://www.youtube.com/watch?v=l99aKKHcxC4
1,000 copies of each fragment are generated by bridge
amplification, creating clusters (30-50 million of clusters).
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Illumina: summary
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Sequenziamento e rilevazione del segnale
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Pyrosequencing (Roche 454)
“Zombie platform” Field guide to next-generation DNA sequencers TRAVIS C. GLENN
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Reversible terminators (Illumina)
Mod. from Metzker 2010
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Reversible terminaotrs (Ion torrent)
No fluorescence! No need to ‘translate’ signal!
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Per una guida comparata alle piattaforme
• http://www.molecularecologist.com/next-gen-fieldguide-2014/