Next Generation Sequencing
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(2) Landmarks in DNA sequencing • 1953 Discovery of DNA double helix structure • 1977 . – A Maxam and W Gilbert "DNA seq by chemical degradation" – F Sanger"DNA sequencing with chain-‐terminating inhibitors" . • 1984 DNA sequence of the Epstein-‐Barr virus, 170 kb • 1987 Applied Biosystems -‐ first automated sequencer • 1991 Sequencing of human genome in Venter's lab • 1996 P. Nyrén and M Ronaghi -‐ pyrosequencing • 2001 A draft sequence of the human genome • 2003 human genome completed • 2004 454 Life Sciences markets first NGS machine 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven -‐ KU Leuven.
(3) Massive parallel sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven-‐KU Leuven.
(4) 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven-‐KU Leuven.
(5) 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven -‐ KU Leuven.
(6) Landmarks in NGS Roche 454. Solexa/Illumina. E. coli (5Mb). SOLiD. Arabidopsis thaliana (157 Mb). 200 K reads 120 bp. 30M reads 35 bp. 100M reads 35 bp. 2005. 2006. 2007 6.
(7) Landmarks in NGS Roche 454. Illumina. SOLiD. Ion torrent. PacBio RS. E. coli (5Mb). Arabidopsis thaliana (157 Mb). 200 K reads 30M reads 100M reads 120 bp 35 bp 35 bp. 2005. 2006. 2007. 2008. 2009. 2010. 7.
(8) DNA Sequencing – the next generation. NGS refers to non-‐Sanger-‐based high-‐throughput DNA sequencing technologies. Millions or billions of DNA strands can be sequenced in parallel.
(9) DNA Sequencing – the next generation • NGS refers to non-‐Sanger-‐based high-‐ throughput DNA sequencing technologies. • NGS technologies constitute various strategies that rely on a combination of – Library/template preparation – Parallel sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven-‐KU Leuven.
(10) DNA Sequencing – the next generation. Sample prep. 10/1/2015. Clonal Amplification. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. Parallel sequencing. 11.
(11) Roche GS FLX 454 & Roche Junior. 454 SEQUENCING. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 12.
(12) 454 sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 13.
(13) 454 sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 14.
(14) 454 sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 15.
(15) Life Technologies SOLiD 5500 Genetic Analyzer. SOLID SEQUENCING. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 16.
(16) SOLiD sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 17.
(17) SOLiD sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 18.
(18) Life Technologies: Ion Proton & Ion PGM. ION TORRENT SEQUENCING. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 19.
(19) Ion Torrent Sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 20.
(20) Ion Torrent Sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 21.
(21) Illumina HiSeq & NextSeq & MiSeq. ILLUMINA (SOLEXA) SEQUENCING. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 22.
(22) Illumina sequencing Library • All sample preparation protocols regardless of the application end with the same product: – Double-‐stranded DNA with the insert to be sequenced flanked by adapters . 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 23.
(23) Illumina library prep. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 24.
(24) Illumina Sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 25.
(25) Illumina Sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 26.
(26) Illumina Sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 27.
(27) Helicos BioSciences: November 15, 2012, bankrupt. HELISCOPE SEQUENCING. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 28.
(28) DNA Sequencing – the next generation. Sample prep. 10/1/2015. Clonal Amplification. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. Parallel sequencing. 29.
(29) Heliscope sequencing. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 30.
(30) Oxford Nanopore Technologies: GridION & MinION. NANOPORE SEQUENCING. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 31.
(31) Oxford Nanopore Technologies: GridION & MiION. NANOPORE SEQUENCING.
(32) Pacific Biosciences PacBio RS II. SMRT SEQUENCING. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 33.
(33) PacBio history • 2010 -‐ PacBio seduced investors with a promise of technology revolution – A whole human genomes for $100 – in about 15 minutes . • 2011 -‐ GC applies for funding for third generation sequencer.
(34) PacBio history • 2012 -‐ None of those predictions came true – Few scientists bought the one-‐ton instrument. – PacBio • market valuation of less than $70 million • technology value of $0. • $600 million of cash down the toilet. . • 2012 – GC gets funding for PacBio! • Oxford Nanopore announced at AGBT.
(35) PacBio history • 2012 – New CEO Mike Hunkapiller @ PacBio . • 2013 – GC installs PacBio – PacBio improved and has a niche • ability to detect structural genetic variations • creating high-‐quality genomes of small organisms like bacteria, viruses, and worms. . – PacBio’s deal with Roche to develop technology for the diagnostic market.
(36) Single Molecule, Real-Time (SMRT®) DNA Sequencing. SMRT® bell. SMRT® Cells. PacBio® RS II.
(37) Template Preparation. Template Template Preparation Preparation. Run Run Design Design. Polymerase Polymeras eBinding Binding. Instrument Instrument Run Run. Primary Primary Analysis Analysis. Secondary Secondary Analysis Analysis. DNA Sample. Fragment DNA. Damage Repair/ End Repair. Ligate adapters. Purify DNA. SMRTbell™ Template preparation can be used to create libraries of various insert sizes from 250 bp to 20,000 bp depending on the needs of the application.. Tertiary Tertiary Analysis Analysis.
(38) Advantages of SMRTbell™ Templates. Key Advantages: • Structurally linear • Topologically circular • Provides sequences of both forward and reverse strands in the same trace.
(39) Base Modification: Discover the Epigenome. Directly observe base modifications using the kinetics of the polymerization reaction during normal sequencing.
(40) Signal Processing and Base Calling Converting pulses of light into DNA bases and kinetic measures. 43.
(41) Understanding Accuracy in SMRT® Sequencing • Single-pass error rate ~11% (predominantly deletions or insertions) • Single Molecule, Real-Time (SMRT®) DNA sequencing achieves highly accurate sequencing results, exceeding 99.999% (Q50) • How is this possible given that single-pass sequence has 1 mistake every 10 nucleotides • Single-pass errors are distributed randomly, which means that they wash out very rapidly upon building consensus..
(42) Sequencing. 45 74.
(43) SMRT® Sequencing Accuracy. Perspective: Understanding SMRT Sequencing Accuracy Data generated with P4-C2 chemistry on PacBio® RS II; Analyzed using Quiver with 2.0.1 SMRT® Analysis.
(44) The PacBio® RS Helps Resolve Genetically Complex Problems. Targeted Comprehensively Sequencing Characterize Genomic Variation. Generate Finished De Novo Assembly Assemblies. Base Modification Automatically detect Detection DNA base modifications. 47.
(45) NGS time line Roche 454. Illumina. SOLiD. Ion torrent. PacBio RS. E. coli (5Mb). Arabidopsis thaliana (157 Mb). 200 K reads 30M reads 100M reads 120 bp 35 bp 35 bp. 2005. 2006. 2007. 2008. 2009. 2010. 2011. 49.
(46) 454. Mb). NGS time line Illumina. SOLiD. Ion torrent. PacBio RS. Arabidopsis thaliana (157 Mb). ads 30M reads 100M reads p 35 bp 35 bp. 2006. 2007. 2008. 2009. 2010. 2011. 50. 2012.
(47) 09. NGS time line Ion torrent. HiSeq 4000. PacBio RS. HiSeq X ten HiSeq2500. 2010. 2011. 51. 2012. PB Sequel. 2013. 2014. 2015. 2016.
(48) NGS Technology: conclusions. 52.
(49) NGS Technology: conclusions. 53.
(50) Summary. 54.
(51) NGS terminology. 55.
(52) NGS as a tool for studying Genome variation and regulation. NGS APPLICATIONS. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 56.
(53) 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ KU Leuven -‐ UZ Leuven. 57.
(54) DNA SEQUENCING. WHOLE GENOME SEQUENCING.
(55) 59.
(56) Copy Number Variations. 60.
(57) Structural Variations. 61.
(58) Whole genome sequencing ì Copy number variation analysis ì Sequencing a genome at 0.1-‐0.3x ì Sequencing a genome at 1-‐3x . ì Structural variation analysis ì Sequencing a genome at 5-‐10x . ì Whole genome re-‐sequencing ì Sequencing a genome at >30x ì yeast, fruit fly, bacterial genomes, human… 62.
(59) DNA SEQUENCING. TARGETED RE-‐SEQUENCING.
(60) Sequencing -‐ the beginning. Random ??? genome sequencing. 10/1/2015. ???. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven-‐KU Leuven. Sanger sequencing • Targeted • 700-‐100 0 bp.
(61) Target enrichment strategies. Random Hybrid genome Capture sequencing. 10/1/2015. PCR based Sanger sequencing. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven-‐KU Leuven.
(62) Target enrichment strategies. 10/1/2015. Jeroen Van Houdt -‐ Genomics Core -‐ UZ Leuven-‐KU Leuven.
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(64) Rapid expression profiling, transcriptome sequencing and small RNA’s. RNA SEQUENCING.
(65) RNA-‐seq.
(66) RNAseq: Gene Expression through sequencing ì. Supports discovery, screening, and profiling . ì. Does not require prior gene knowledge or annotation . ì. Unique combination of Qualitative and quantitative measurement . ì. Digital counts vs analog intensities . ì. Increased dynamic range and sensitivity . ì. No probes or primers . ì. Any species -‐ Even when reference genome not available . ì. Analyze gene expression.
(67) RNAseq: summary ì. Counting or Profiling ì. ì. Studying Alternative Splicing or quantifying cSNPs for most transcripts ì. ì. 10 million total reads of 35 bp length from poly-‐A selected RNA will give performance better than any microarray Deeper profiling of 50 to 100 million reads, with read lengths of 50 to 100 bps, from poly-‐A selected RNA using mRNA-‐Seq assay . Complete Annotation of an entirely New Transcriptome ì ì ì. ~500 Million reads of 100 bp read length from multiple tissues Normalized stranded mRNA-‐Seq & ncRNAs Small RNA-‐Seq for microRNAs.
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