Third Generation Sequencing By Mohammad Hasan Samiee Aref Medical Genetics Laboratory of Dr. Zeinali
History of DNA sequencing 1953 : Discovery of DNA structure by Watson and Crick 1973 : First sequence of 24 bp published 1977 : Sanger sequencing method published 1980 : Nobel Prize Wally Gilbert and Fred Sanger 1982 : Genbank started 1983 : Development of PCR 1987 : 1 st automated sequencer : Applied Biosystems Prism 373
History of DNA sequencing 1996 : Capillary sequencer: ABI 310 1998 : Genome of Caenorhabditis elegans sequenced 2000 : Human genome sequenced 2005 : 1 st 454 Life Sciences Next Generation Sequencing system: GS 20 System 2006 : 1 st Solexa Next Generation Sequencer: Genome Analyzer 2007 : 1 st Applied Biosystems Next Generation Sequencer: SOLiD 2009 : 1 st Helicos single molecule sequencer: Helicos Genetic Analyser System 2011 : 1 st Ion Torrent Next Generation Sequencer: PGM 2011 : 1 st Pacific Biosciences single molecule sequencer: PacBio RS Systems 2012 : Oxford Nanopore Technologies demonstrates ultra long single molecule reads
Different platforms 454 Sequencing / Roche GS Junior System GS FLX+ System illumina (Solexa) HiSeq System Genome analyzer IIx MiSeq Applied Biosystems - Life Technologies SOLiD 5500 System SOLiD 5500xl System Ion Torrent - Life Technologies Personal Genome Machine (PGM) Proton Next Generation Sequencing Amplified Single Molecule Sequencing
Different platforms Helicos Helicos Genetic Analysis System Pacific Biosciences PacBio RS Oxford Nanopore Technologies GridION System Third Generation Sequencing Next Next Generation Sequencing Single Molecule Sequencing
Workflow Library preparation Emulsion PCR Polony PCR on a slide Semiconductor sequencing (Ion Torrent) Pyrosequencing (454) Sequencing by ligation (SOLiD) Sequencing by ligation (SOLiD) Reversible terminator sequencing (Illumina)
Workflow (Library preparation) Good fragments:
Workflow Library preparation Emulsion PCR Polony PCR on a slide Semiconductor sequencing (Ion Torrent) Pyrosequencing (454) Sequencing by ligation (SOLiD) Sequencing by ligation (SOLiD) Reversible terminator sequencing (Illumina)
Workflow (Emulsion PCR)
Workflow Library preparation Emulsion PCR Polony PCR on a slide Semiconductor sequencing (Ion Torrent) Pyrosequencing (454) Sequencing by ligation (SOLiD) Sequencing by ligation (SOLiD) Reversible terminator sequencing (Illumina)
Workflow ( Polony PCR Bridge amplification: illumina)
Workflow ( Polony PCR Bridge amplification: illumina)
Workflow ( Polony PCR Bridge amplification: illumina)
Different platforms (illumina)
Different platforms (illumina) HiSeq HiScanSQ Genome Analyzer llx MiSeq Read Length 100 bp 100 bp 150 bp 250 bp Throughput 600 Gb 150 Gb 95 Gb 6 Gb Reads per run 3,000,000,000 750,000,000 320,000,000 12,000,000 Accuracy 99,9 % 99,9 % 99,9 % 99,9 % Run Time 11 days 8 days 14 days 20 35 hours Workflow: Library preparation Bridge amplification Reversible termination sequencing
Different platforms (Ion Torrent) PGM (Personal Genome Machine) Proton
Different platforms (Ion Torrent) PGM Proton Read Length 200 bp 200 bp Throughput 20 Mb 1 Gb 10 100 Gb Reads per run 11,000,000 660,000,000 Accuracy 99 % 99 % Run Time 4.5 hours 4.5 hours Workflow: Library preparation Emulsion PCR Semiconductor sequencing
Analysis (Sanger Sequencing)
Analysis (Sanger Sequencing)
Analysis (Next Generation Sequencing)
Analysis (Next Generation Sequencing)
Analysis (Next Generation Sequencing) Same dataset, different parameters
Different platforms (Third generation) Helicos Genetic Analysis System (Helicos BioSciences Corporation) Helicos Read Length 35 bp Throughput 35 Gb Reads per run 600,000,000 1,000,000,000 Accuracy 97% Run Time 8 days Workflow: Library preparation Sequencing
Different platforms (Third generation) Advantages of Single molecule sequencing: Less sample preparation (no PCR) No amplification -no PCR errors -fewer contamination issues -no GC-bias -analyze every sample (unpcrable / unclonable) -analyze low quality DNA (museum, archeological, forensics samples) Absolute quantification Sequence RNA directly
Different platforms (Third generation) Pacbio RS (Pacific Biosciences) Pacbio RS Read Length 3000 15,000 bp Throughput 1 Gb Reads per run 70,000 Accuracy 95% Run Time 30 minutes Workflow: Library preparation Sequencing
Different platforms (Oxford Nanopore) MinION PromethION GridION
MinION
MinION Nanopore Array of Microscaffolds Array Chip ASIC (Application Specific Integrated Circuit)
MinION The MinION MkI flow cell
MinION
PromethION
Specification MinION PromethION Number of channels available for sequencing Sample input Requirement PCR Free Flow cell input volume Mk 1 MinION Single PromethION Flow Cell PromethION (48 Flow Cells) Up to 512 Up to 3,000 Up to 144,000 10pg - 1μg 10pg - 1μg 10pg - 1μg 50-160μl 35μl per sample well (4 wells in a flow cell) 35µl per sample well (192 wells in a PromethION)
Specification MinION PromethION Sample preparation time 1D Sample preparation time 2D Mk 1 MinION Single PromethION Flow Cell PromethION (48 Flow Cells) 10 minutes 10 minutes 10 minutes 90 minutes 90 minutes 90 minutes Run time 1 minute - 48 hours 1 minute - 48 hours 1 minute - 48 hours Flow cell lifetime ~72hrs >= 72hrs >= 72hrs
Specification MinION PromethION Number of reads at 10Kb at standard speed (250bps) Number of reads at 10kb in Fast Mode (500bps) Read Length Mk 1 MinION Single PromethION Flow Cell PromethION (48 Flow Cells) Up to 2.2M Up to 13M Up to 625M Up to 4.4M Up to 26M Up to 1250M Longest reported between 230-300 Kilobases (1D) Longest reported between 230-300 Kilobases (1D) Longest reported between 230-300 Kilobases (1D)
Specification MinION PromethION 1D Yield at 250 bps in 48 hours 1D Yield at 500 bps in 48 hours Mk 1 MinION Single PromethION Flow Cell PromethION (48 Flow Cells) Up to 21 Gb Up to 128 Gb Up to 6 Tb Up to 42 Gb Up to 256 Gb Up to 12 Tb Base calling accuracy Up to 99% Up to 99% Up to 99% Modified Base Detection Yes Yes Yes
Specification MinION PromethION Mk 1 MinION Single PromethION Flow Cell PromethION (48 Flow Cells) Data Analysis Local offline/online On Unit / On line On Unit / On line Power requirement USB 3 (1 watt) < 20 watts ~ 1kW Potential raw data requirements (Bytes per second) up to 5MBps up to 30MBps up to 1.44GBps Weight 87g 40 Kg 40 Kg
Specification MinION PromethION Mk 1 MinION Single PromethION Flow Cell PromethION (48 Flow Cells) Reagent cost per run $ 99 POA POA Flow Cell Cost (depending on order type and volume) $270 - $900 POA POA Instrument Access Fee Starter kit (includes MinION and all materials for 2 runs) $1000 No instrument cost - $75K deposit to be called off against consumable purchases No instrument cost - $75K deposit to be called off against consumable purchases
GridION
SmidgION
Fields of use Sensing of biological molecules
Fields of use For research or industrial purposes
Fields of use In a variety of areas
Fields of use On any sample of interest
Fields of use Protein analysis Direct, electronic analysis of proteins by combining nanopores with aptamers
Fields of use RNA
Fields of use MicroRNA
Fields of use MicroRNA
Fields of use MicroRNA
Publications 56 publications (from August 2014 until now)
Thank you for kind attention