Next Generation Sequencing. Jeroen Van Houdt - Leuven 13/10/2017
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1 Next Generation Sequencing Jeroen Van Houdt - Leuven 13/10/2017
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 Life Sciences markets first NGS machine Jeroen Van Houdt - Genomics Core - UZ Leuven - KU Leuven
3 Massive parallel sequencing Jeroen Van Houdt - Genomics Core - UZ Leuven-KU Leuven
4
5 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 Jeroen Van Houdt - Genomics Core - UZ Leuven-KU Leuven
6 Landmarks in NGS Roche 454 Solexa/Illumina SOLiD E. coli (5Mb) Arabidopsis thaliana (157 Mb) 200 K reads 120 bp 30M reads 35 bp 100M reads 35 bp
7 Landmarks in NGS Roche 454 Illumina SOLiD Ion torrent PacBio RS E. coli (5Mb) Arabidopsis thaliana (157 Mb) 200 K reads 120 bp 30M reads 35 bp 100M reads 35 bp
8 Landmarks in NGS Ion torrent PacBio RS HiSeq 4000 HiSeq2500 HiSeq X ten 1000 $ genome PB Sequel NovaSeq 6000 Goal: a 100 $ genome
9 Next Generation Sequencing Sample prep Clonal Amplification Parallel sequencing Jeroen Van Houdt - Genomics Core - UZ Leuven-KU Leuven
10 454 sequencing Roche GS FLX 454 & Roche Junior Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 11
11 454 sequencing Clonal Amplification Parallel sequencing 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 12
12 454 sequencing 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 13
13 454 sequencing 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 14
14 Solid sequencing Life Technologies SOLiD 5500 Genetic Analyzer 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 15
15 SOLiD sequencing 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 16
16 SOLiD sequencing 20 $ / Gb 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 17
17 Ion torrent sequencing Life Technologies: Ion Proton & Ion PGM Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 18
18 Ion Torrent Sequencing Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 21
19 Ion Torrent Sequencing Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 22
20 Illumina (solexa) sequencing Illumina MiSeq, NextSeq 500, HiSeq2500, HiSeq4000 & NovaSeq /13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 23
21 Sample prep Clonal Amplification Parallel sequencing Template preparation Produce a non-biased source of nucleic acid material from the genome Current methods: Sonication randomly breaking genomic DNA into smaller sizes Enzymatic digestion Tagmentation Jeroen Van Houdt - Genomics Core - UZ Leuven-KU Leuven
22 Sample prep Clonal Amplification Parallel sequencing 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 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 25
23 Sample prep Clonal Amplification Parallel sequencing Illumina library prep 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 26
24 Sample prep Clonal Amplification Parallel sequencing Illumina library prep 27
25 Sample prep Clonal Amplification Parallel sequencing NGS Illumina Clustering
26 Sample prep Clonal Amplification Parallel sequencing Single DNA libraries are hybridized to primer lawn Adapter sequence Bound libraries then extended by polymerases Surface of flow cell coated with a lawn of oligo pairs 3 extension Gel size selection, if needed
27 Sample prep Clonal Amplification Parallel sequencing NOTE: Single molecules bind to flow cell in a random pattern Gel size selection, if needed
28 Sample prep Clonal Amplification Parallel sequencing Double-stranded molecule is denatured Original template Newly synthesized strand Original template washed away discard Newly synthesized strand is covalently attached to flow cell surface Gel size selection, if needed
29 Sample prep Clonal Amplification Parallel sequencing Single-stranded molecule flips over and forms a bridge by hybridizing to adjacent, complementary primer Hybridized primer is extended by polymerases Gel size selection, if needed
30 Sample prep Clonal Amplification Parallel sequencing Double-stranded bridge is formed Gel size selection, if needed
31 Sample prep Clonal Amplification Parallel sequencing Double-stranded bridge is denatured Result: Two copies of covalently bound single-stranded templates Gel size selection, if needed
32 Sample prep Clonal Amplification Parallel sequencing Single-stranded molecules flip over to hybridize to adjacent primers Hybridized primer is extended by polymerase Gel size selection, if needed
33 Sample prep Clonal Amplification Parallel sequencing Bridge amplification cycle repeated until multiple bridges are formed Gel size selection, if needed
34 Sample prep Clonal Amplification Parallel sequencing dsdna bridges are denatured Gel size selection, if needed
35 Sample prep Clonal Amplification Parallel sequencing Reverse strands cleaved and washed away, leaving a cluster with forward strands only Gel size selection, if needed
36 Sample prep Clonal Amplification Parallel sequencing Free 3 ends are blocked to prevent unwanted DNA priming Gel size selection, if needed
37 Sample prep Clonal Amplification Parallel sequencing Sequencing primer is hybridized to adapter sequence Sequencing primer Gel size selection, if needed
38 Sample prep Clonal Amplification Parallel sequencing Flow Cell - Clusterdensity 20pM 10pM 5pM 1pM
39 Sample prep Clonal Amplification Parallel sequencing Flow Cell - Clusterdensity Non-Patterned FC Only a small area of the signal is considered to avoid cluster overlap Cluster A Cluster B threshold Values below threshold are not considered Patterned FC
40 Sample prep Clonal Amplification Parallel sequencing Patterned flow cell Jeroen Van Houdt 43
41 Sample prep Clonal Amplification Parallel sequencing Duplicates Jeroen Van Houdt 44
42 Sample prep Clonal Amplification Parallel sequencing MiSeq NextSeq 500 HiSeq 2500 HiSeq 4000 NovaSeq 6000 Jeroen Van Houdt 45
43 Sample prep Clonal Amplification Parallel sequencing NGS Illumina Sequencing Sequencing By Synthesis (SBS)
44 Sample prep Clonal Amplification Parallel sequencing NGS Illumina Sequencing: Imaging Four color system Two color system
45 Sample prep Clonal Amplification Parallel sequencing NGS Illumina Sequencing Single index sequencing Gel size selection, if needed 1 2 Paired End Turnaround 3 Gel size selection, if needed
46 Sample prep Clonal Amplification Parallel sequencing NGS Illumina Sequencing Dual index sequencing Gel size selection, if needed Paired End Turnaround 4 Gel size selection, if needed
47 Sample prep Clonal Amplification Parallel sequencing NGS Illumina Sequencing Dual index sequencing NextSeq 500 Gel size selection, if needed Index Read 2 (i5) i7 Index Paired End Turnaround Index read 3 i5 Index i5 Index Seq Primer (SBS3rev/BP13) 4 Gel size selection, if needed
48 Illumina Sequencing 100 Microns 51
49 MiSeq NextSeq 500 HiSeq 2500 HiSeq 4000 NovaSeq 6000 Jeroen Van Houdt 52
50 NGS Illumina Sequencing: summary Sample prep Clonal Amplification Parallel sequencing 53
51 NGS Illumina Sequencing: terminology Single-end vs Paired-end 100 Microns Gel size selection, if needed
52 Qiagen GeneReader NGS Jeroen Van Houdt 55
53 HeliScope Sequencing Helicos BioSciences: November 15, 2012, bankrupt 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 56
54 NGS: third generation Sample prep Clonal Amplification Parallel sequencing Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven
55 Heliscope sequencing 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 58
56 Heliscope SeqLL, the startup formed in 2013 to revive the single-molecule sequencing technology originally developed by Helicos BioSciences Early-access program for the system at end 2016, Aims to start selling commercially in mid Jeroen Van Houdt 59
57 NANOPORE sequencing Oxford Nanopore Technologies: MinION, GridION, PromethION & SmidgION Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 60
58 Oxford Nanopore sequencing 61
59
60 Nanopore sequencing MinION GridION PromethIOM SmidgION Jeroen Van Houdt 65
61 Oxford Nanopore sequencing 66
62 Roche nanopore sequencing (Genia's technology) Dec 2016 the company plans to focus its efforts on developing Genia's nanopore technology for clinical use, Genia's technology has "progressed very well" and that Roche would announce a timeline for the commercial launch of the system next year. Roche's "development focus from a sequencing perspective will be very much on that platform now," Gunn said. "Ultimately, our objective is to have an end-to-end solution within the clinical sequencing space." October 13, 2017 Jeroen Van Houdt 67
63 Roche nanopore sequencing (Genia's technology) 68
64 SMRT Sequencing Pacific Biosciences PacBio RS II & Sequel 10/13/17 Jeroen Van Houdt - Genomics Core - KU Leuven - UZ Leuven 69
65 PacBio history PacBio seduced investors with a promise of technology revolution A whole human genomes for $100 in about 15 minutes GC applies for funding for third generation sequencer
66 PacBio history 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 GC gets funding for PacBio! Oxford Nanopore announced at AGBT
67 PacBio history 2012 New CEO Mike 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
68 Single Molecule, Real-Time (SMRT ) DNA Sequencing SMRT bell SMRT Cells PacBio RS II
69 Advantages of SMRTbell Templates Key Advantages: Structurally linear Topologically circular Provides sequences of both forward and reverse strands in the same trace
70 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 % (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.
71 SMRT Sequencing Accuracy Data generated with P4-C2 chemistry on PacBio RS II; Analyzed using Quiver with SMRT Analysis
72 Benefits of SMRT Sequencing Produce reads with average lengths of 3000 to 5000, with longest reads over 20,000 base pairs Greater than % (QV 50) accurate sequencing results Sensitivity to detect minor variants at frequency less than 0.1% Detect broad spectrum of base modification events in the same sequencing run that reads canonical base sequence No amplification bias and least GC bias for improved coverage uniformity Shortest run time 83
73 SMRT sequencing Targeted Sequencing De Novo Assembly Base Modification Detection Comprehensively Characterize Genomic Variation Generate Finished Assemblies Automatically detect DNA base modifications 84
74 Summary Clonal amplification Roche 454 Illumina NovaSeq/HiSeq/NextSeq/MiSeq Life SOLiD Life Ion Torrent / PGM Single molecule sequencing Helicos BioSciences HeliScope -> now SeqLL Pacific Biosciences PacBio RS PacBio Sequel Oxford Nanopore 10/13/17 Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
75 Summary In solution emulsion PCR (empcr) Roche 454 Life SOLiD Solid phase Bridge PCR Illumina HiSeq Life SOLiD - wildfire 10/13/17 Jeroen Van Houdt - Genomics Core - UZ Leuven- KU Leuven
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