the past, present, and future of dna sequencing*. What NGS Consists of Next generation technologies for sequencing is combination of strategies for • template preparation • sequencing and imaging • genome alignment • assembly methods 9. The $1,000 dollar genome – A free PowerPoint PPT presentation (displayed as a Flash slide show) on PowerShow.com - id: 3bce93-N2I5Y 6 Part # 15045845_Rev.D FOR RESEARCH USE ONLY Library Preparation dr. a.c.m. Welcome to Next-Generation Sequencing 3 a. These techniques allow the analysis of around 500 nucleotides of DNA a time. The turn-around time of the second generation sequencing technology to complete a human genome project can just be one week, while that using the first generation sequencing technology to achieve the same goal is three years. This From slab gels to capillary sequencing and second-generation sequencing technologies, there has been a more than a million-fold improvement in the rate of sequence generation over this time scale. An illustrated glossary and a video overview of next-generation sequencing are available at NEJM.org The New England Journal of Medicine Downloaded from nejm.org at HOUSTON ACADEMY OF MEDICINE on October 4, 2018. Offered by Johns Hopkins University. The Basics of NGS Chemistry 4 c. Advances in Sequencing Technology 5 Paired-End Sequencing 5 Tunable Coverage and Unlimited Dynamic Range 6 Multiplexing 6 Advances in Library Preparation 7 Flexible, Scalable Instrumentation 7 II. 2001: Human Genome Project 2.7G$, 11 years 2007: 454 1M$, 3 months 2008: ABI SOLiD 60K$, 2 weeks 2001: Celera 100M$, 3 years 2009: Illumina, Helicos 40-50K$ 2000 Sequencing the Human Genome 10 8 6 Log10(price) 2010: 7K$, a few days 4 2 2014: 1000$, 24 hrs? • AmpliconLibrary Preparation, eg.for detection of rare variants. The high demand for low-cost sequencing has driven the development of high-throughput sequencing which produce thousands or millions of sequences at once. Presentation Summary : The extracted DNA was amplified and subjected to Next Generation Sequencing (NGS) using the Ion Ampliseq Cancer Panel hotspot v2 on the Ion Torrent Personal Introduction to Next Generation Sequencing. In the previous lesson on basic DNA sequencing techniques, we covered a variety of sequencing methods that were used from the mid 80's to the early 2000's.. If you continue browsing the site, you agree to the use of cookies on this website. TG..GT TC..CC AC..GC CG..CA TT..TC TG..AC AC..GC GA..GC CT..TG AC..GC GT..GC AC..GC AA..GC AT..AT TT..CC Short DNA sequences ACGTGGTAACGTATACAC TAGGCCATAGTAATGGCG CACCCTTAGTGGCGTATACATA… ACGTGGTAATGGCGTATACACCCTTAGGCCATA ACGTGACCGGTACTGGTAACGTACACCTACGTGACCGGTACTGGTAACGTACGCCTACGTGACCGGTACTGGTAACGTATACACGTGACCGGTACTGGTAACGTACACCTACGTGACCGGTACTGGTAACGTACGCCTACGTGACCGGTACTGGTAACGTATACCTCT... Sequenced genome Sanger can do whole genomes (painfully) Genome Short fragments of DNA 20. Course - Introduction to Next-Generation Sequencing. But for now, know that NGS is an umbrella descriptor for sequencing millions of DNA data points at the same time. Next-generation sequencing (NGS) technologies using DNA, RNA, or methylation sequencing have impacted enormously on the life sciences. Slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Introduction to next generation sequencing 1. Clipping is a handy way to collect important slides you want to go back to later. He joined the university in 2015. biotechnology. Keywords SMRT Sequencing;PacBio;Long read sequencing;Third generation sequencing;Single Molecule, Real-Time;Pacific Biosciences;PacBio Systems;Next gen sequencing;Next generation sequencing;How it Works;Poster The Millennial Generation: The Next Generation in College Enrollment - . Table of Contents I. Automated sequencing machines use 4 colors, so they can read all 4 bases at once. Presented by. You can change your ad preferences anytime. Get an introduction to this technique from the video. Remove second strand Attach to library beads via “B” adapter at calculated concentration to yield a single template molecule per library bead 454: Shotgun Library Preparation Proceed to emPCR Images from: https://www.roche-applied-science.com/, 454: AmpliconLibrary Preparation • Target amplicon of 200-500 bp • 200 bp for uni-direction reads • 500 bp requires bi-directional reads • Amplify using fusion primers that include template specific primer and primers A and B • Purify and quantify • Proceed to emPCR, From Debbie Nickerson, Department of Genome Sciences, University of Washington, http://tinyurl.com/6zbzh4, Illumina (Solexa) Applications Resequencing • Characterise different related species or strains Transcriptome analysis • No chip/array required! 5 Part # 15045845_Rev.D FOR RESEARCH USE ONLY Library Prep is Critical for Successful Sequencing The aim of library prep is to obtain nucleic acid fragments with adapters attached on both ends Dual Index Library shown. Template preparation As even most sensitive imaging technique is not able to detect single molecule, amplification of templates is inevitable. Next-generation sequencing (NGS) technologies using DNA, RNA, or methylation sequencing have impacted enormously on the life sciences. • Data Analysis Pipeline • Sequence QC and preprocessing • Obtaining and preparing reference • Sequence … In this method, DNA fragments are hybridized in solution to sequence-specific capture Nucleic acid sequencing is a method for determining the exact order of nucleotides present in a given DNA or RNA molecule. The resulting cDNA can then be sequenced using any 2010 name sequence quality scores x 100s of millions (slide courtesy of Ben Langmead) 44. Nat Rev Genet. 19 may 2014 jim evans, Ion Torrent and Minion - . Next-generation sequencing generates masses of DNA sequencing data, and is both less expensive and less time-consuming than traditional Sanger sequencing. The practicals are the subparts of the assignment Write up in more or less ‘paper format’ or in standard prac format Due end of day on 16 March (Sunday). Sequencing large pieces of DNA VI. At its inception massively parallel sequencing was ill suited for the task of sequencing the human genome. Medical Director of Molecular Genetics and Genomics . Department Life Sciences. SMRT Sequencing allows you to accelerate your science with the complete range of PacBio applications to produce data you can trust. Next Generation Sequencing – Whole Exome Sequencing (WES) The exome accounts for only 1.5% of the human genome, and yet includes 85% of all disease-causing mutations (10). Dr Vladimir Teif is a Lecturer in the School of Life Sciences at the University of Essex, an author of more than 40 research publications. Since completion of the first human genome sequence, demand for cheaper and faster sequencing methods has increased greatly. About the assignment. 2005 2010 Year, Sanger vs NGS • ‘Sanger sequencing’ has been the only DNA sequencing method for 30 years but… • NGS has the ability to process millions of sequence reads in parallel rather than 96 at a time (at a smallfractionof the cost), High Throughput Sequencing Massively parallel sequencing • Sequencing millions of molecules in parallel • Do not need prior knowledge of what you’re sequencing We will work with Illumina data only, 454 = Paradigm Shift • Standard ABI “Sanger” sequencing • 96 samples/day • Read length ~650 bp= 450,000 bases • 454 • ~400,000 different templates (reads)/day • Read length ~250 bp • Total = 100,000,000 bases of sequence data, Solexa ups the Game • Solexa (Illumina GA) • 60,000,000 different sequence templates (yes that is an insane 60 million reads) • originally 36 bp read length (much longer now) • 4 billion bases of DNA per run (3 days), Some NGS milestones • 454 Life Sciences/Roche • Genome Sequencer FLX: currently produces 400-600 million bases per day per machine • Published 1 million bases of Neanderthal DNA in 2006 • May 2007 published complete genome of James Watson (3.2 billion bases ~20x coverage) • Solexa/Illumina • 10 GB per machine/week • May 2008 published complete genomes for 3 hapmap subjects (14x coverage) • ABI SOLID • 20 GB per machine/week, The general NGS workflow Random shearing of the DNA Adding adaptors and barcodes Size selection Amplification Sequencing, It’s all about nanotechnology • Each system works differently, but they are all based on a similar principals: • Shear target DNA into small pieces • bind individual DNA molecules to a solid surface, • amplify each molecule into a cluster • copy one base at a time and detect different signals for A, C, T, & G bases • requires very precise high-resolution imaging of tiny features • (Solexa has 800 images @ 4 megapixels each), 454 Sequencing Overview • Prepare library of single stranded DNA, 200-500 bp long and ligate adapters • Perform emulsion PCR, amplifying a single DNA template molecule in each microreactor (bead). And right now, the sequencing technology can be divided into next-generation sequencing and third generation sequencing. gaston, The Millennial Generation: The Next Generation in College Enrollment - . Next-Generation Sequencing. Liquid Biopsy Overview, Challenges and New Solutions: Liquid Biopsy Series Pa... Owncloud, la alternativa de fuentes abiertas a Dropbox, No public clipboards found for this slide, Introduction to next generation sequencing, JSS University (Jagadguru Sri Shivarathreeshwara University),Mysore. Brief history of sequencing II. Sanger dideoxy method for sequencing III. Select for “A” – “B” fragments. DNA sequencing: methods I. In the past decade, the use of nucleic acid sequencing has increased exponentially as the ability to sequence has become accessible to research and clinical labs all over the world. • Sequence all clonally amplified sample fragments in parallel using pyrosequencing technology • Analyze sequence results • CLEAN data • Align overlapping sequence of individual reads to define contigs (Shotgun) • Order and orient contigs, create scaffolds (Paired End) • Identify variants (Amplicon) • Determine gene expression patterns (Transcriptome), Emulsion Based Clonal Amplification A + PCR Reagents + Emulsion Oil B Micro-reactors Mix DNA Library & capture beads (limited dilution) Create “Water-in-oil” emulsion Adapter carrying library DNA “Break micro-reactors” Isolate DNA containing beads Perform emulsion PCR • Generation of millions of clonally amplified sequencing templates on each bead From: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt ), Load Enzyme Beads Load beads into PicoTiter™Plate 44 μm Depositing DNA Beads into the PicoTiter™Plate Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt ), PicoTiterPlate Wells Reagent Flow PhotonsGenerated are Captured by Camera Sequencing By Synthesis Reagent flow and image capture Sequencing Image Created Adapted from: Roche 454 James Grabeau 2007 (www.lsbi.mafes.msstate.edu/Roche%20454%20James%20Grabau%202007.ppt ), FLX Sequencing Reaction www.roche-applied-science.com. Introduction to Next Generation Sequencing. ARUP Laboratories . gaston college november 1, 2010. terri manning, Instructional Sequencing - . Introduction to Next-Generation Sequencing (NGS) CGEpi Winter school 2017 13.02.2017 Aarif Mohamed Nazeer Batcha & Guokun Zhang 10.jpg About the assignment. Introduction - Next Generation Sequencing 9.00-9.30 Introduction to course & Pre-test (Lecture slides) Simon Rasmussen 9.30-10.00 Introduction to NGS (Lecture slides) Simon Rasmussen 10.00-10.45 2nd and 3rd generation NGS Technologies (Lecture slides) Simon Rasmussen 10.45-11.00 Break 11.00-12.00 accomplishments, challenges and opportunities. An Introduction to Next-Generation Sequencing Technology . 1. applications for next-generation sequencing. DNA sequencing “the technology lecture” - . Nucleic acid sequencing is a method for determining the exact order of nucleotides present in a given DNA or RNA molecule. Basically, in NGS millions of small pieces of the genome are sequenced at the same time, rather than trying to read it all in order. Department Life Sciences. nucleic acid sequencing for the subsequent two and a half decades (Sanger et al., 1977). This course introduces you to the basic biology of modern genomics and the experimental tools that we use to measure it. Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies. terri manning bobbie everett cheryl roberts. dan, Computational assembly for prokaryotic sequencing projects - . understand how dna, The Millennial Generation: The Next Generation in College Enrollment - . sequencing sequencing element design max and min-delay clock skew time, Sequencing, Sequence Alignment & Software - . I'm a Postdoc from the Research Group for Genomic Epidemiology at DTU Food. Part 1: Introduction. nanopore, Ontology Generation and Applications - . Offered by Johns Hopkins University. NGS is the choice for large-scale genomic and transcriptomic sequencing because of the high-throughput production and outputs of sequencing data in the gigabase range per instrument run and the lower cost compared to the traditional Sanger first-generation … 2 Illumina sequencing systems can deliver data output ranging from 300 kilobases up to multiple terabases in a single run, depending on instrument type and configuration. Introduction to Next Generation Sequencing (NGS) Andrew Parrish Exeter, 2 nd November 2017 Create stunning presentation online in just 3 steps. Duration 2 days. The Introduction Of High Throughput, Next Generation Sequencing (ngs) In 2005 Heralded A 419578 PPT. Part 1: Introduction. Presentation Summary : The extracted DNA was amplified and subjected to Next Generation Sequencing (NGS) using the Ion Ampliseq Cancer Panel hotspot v2 on the Ion Torrent Personal Capillary electrophoresis (CE)-based sequencing has enabled scientists to elucidate genetic information from almost any organism or biological system. Introduction to Next-Generation Sequencing (NGS) CGEpi Winter school 2017 13.02.2017 Aarif Mohamed Nazeer Batcha & Guokun Zhang 10.jpg http://cancergenome.nih.gov/newsevents/multimedialibrary/images/CancerBiology, Front matter: Central Dogma of Molecular Biology Reverse Transcription, Why does that matter? Finding the sequence of a DNA/ RNA molecule What can we sequence? Welcome to Next-Generation Sequencing a. Cluster Generation Sequencing. With the advent of capillary electrophoresis (CE)-based sequencing in 1977, scientists gained the ability to sequence the full genome of any species in a reliable, reproducible manner. part 1: chemistry, instrumentation and data analysis part 2: large-scale, Chapter 20 - . Academia.edu is a platform for academics to share research papers. Presentation Summary : The introduction of high-throughput, next-generation sequencing (NGS) in 2005 heralded a critical and transformative step in the history of DNA sequencing. sequencing Year 2005 Kilobases per day per machine Figure 3 | Improvements in the rate of DNA sequencing over the past 30 years and into the future. Presented by. DNA sequencing: methods I. See our Privacy Policy and User Agreement for details. DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA.It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. An Introduction to Illumina Next-Generation Sequencing Technology for Microbiologists Deciphering DNA sequences is essential for virtually all branches of biological research. DNA sequencing exploits the physicochemical properties of DNA and the enzymes involved in its replication, The DNA molecule • The two strands of DNA are different • One is called the sense strand and it is the plan to make a protein • The other strand is the antisense strand, Connecting the DNA molecule • The two strands of DNA are said to be antiparallel • One strand is oriented in a 5’ to 3’ direction • The other strand is oriented in the opposite 3’ to 5’ direction 5’ 3’ antisense sense 3’ 5’, Introns and Exons • Introns– non-codingsequences in the DNA that are NOT used to make to make a protein • Exons–coding sequences in the DNA that are expressed or used to make mRNA and ultimately are used to make a protein, Sanger Method Fred Sanger, 1958 Was originally a protein chemist Made his first mark in sequencing proteins Made his second mark in sequencing RNA 1980 dideoxy sequencing, Sanger Method: Dideoxy Chain Termination 300-500 bases, Capillary Method - Fluorescent Dyes 800-1000 bases. At first this technology was cost prohibitive for all but a few academic and industry institutions, however, as the instrumentation and technology continues to … Introduction of Next Generation Sequencing into Clinical Diagnostics Rong Mao, MD Associate Professor of Pathology . The teachers are experienced and have much knowledge of the topics. Presentation Title: Next Generation Sequencing. Sanger sequencing was used in the Human Genome Project to determine the sequences of relatively small fragments of human DNA (900 bp or less). Duration 2 days. Next Generation Sequencing, It And The Clinical Practice PPT. With so much data at their fingertips, scientists can learn more about food and environmental microbial communities than ever before. a, CSER Clinical Sequencing Exploratory Research - . what is sequencing? We'll introduce the Central Dogma of Molecular Biology and cover how next-generation sequencing can be used to measure DNA, RNA, and epigenetic patterns. This demand has driven the develop-ment of second-generation sequencing methods, or next-generation sequencing (NGS). It is an excellent course and I can highly recommend it. This video is part one of the Next Generation Sequencing miniseries. Introduction The first critical step of controlling a plant disease is a rapid diagnosis that accurately identifies the causal agent(s). Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies. You get exactly what you are promised: a theoretical overview of Next-Generation Sequencing. Although these techniques allowed us to sequence the first human genome, they were too costly and time-intensive. relatively low cost ‘next generation’ sequencing. Automated Sequencing • Leroy Hood developed fluorescent color labels for the 4 terminator nucleotide bases (late 80s). Course - Introduction to Next-Generation Sequencing. You'll also get an introduction to the key concepts in computing … sequencing of the human genome was completed by 2007( 3 million, Lecture 11: Sequential Circuit Design - . sanger dideoxy method for sequencing iii. Introduction to Next Generation Sequencing Analysis. The Basics of NGS Chemistry 4 c. Advances in Sequencing Technology 5 Paired-End Sequencing 5 Tunable Coverage and Unlimited Dynamic Range 6 Multiplexing 6 Advances in Library Preparation 7 Flexible, Scalable Instrumentation 7 II. what strategies are available to, Some new sequencing technologies - . dideoxy sequencing (sanger sequencing, chain terminator method). -2001 The HGP consortium publishes its working draft in Nature (15 February), and Celera publishes its draft in Science (16 February). ... Next generation Sequencing• Improvements in enzymes, chemistry and image analysis, mature by the middle of last decade dramatically increased sequencing capabilities.• ... Ngs ppt Archa Dave. The study objectives are to present the Next-Generation Sequencing Informatics development in North America, Europe, China, Japan, Southeast Asia, … – 3 – I. Next-Generation Sequencing to Diagnose Suspected Genetic Disorders David R. Adams, M.D., Ph.D., and Christine M. Eng, M.D. A guide to the technology, analysis workflows, tools, and resources for next generation sequencing data analysis. A RNA fraction of interest is selected, fragmented and reverse transcribed. Target audience PGR Students in Life Sciences, Maths and CSEE. lee katz, ph.d. bioinformatician, enteric diseases, 第二章 下一代网络与 MPLS Chapter 2 Next Generation Network and MPLS - . Welcome to Next-Generation Sequencing a. Next-generation sequencing generates masses of DNA sequencing data, and is both less expensive and less time-consuming than traditional Sanger sequencing. Presentation Summary : Figure 5 | Overview of RNA-Seq. The workshop provided an introduction to NGS and details related to the emerging laboratory approach. See our User Agreement and Privacy Policy. Sequencing technologies - the next generation. – 3 – I. It is an excellent course and I can highly recommend it. Introduction to Next Generation Sequencing (NGS) In Week 3 we introduced you to the traditional techniques used for genetic testing: PCR and Sanger sequencing. Gene mapping and DNA markers Afsath Begum. Looks like you’ve clipped this slide to already. The first major foray into DNA sequencing was the Human Genome Project, a … single molecule array for genotyping—solexa. lushan wang, shandong university. fong, ceng professor of computer engineering school of computing and, DNA sequencing: methods - . 电控学院 电子工程学科部, The Millennial Generation: The Next Generation in College Enrollment - . Next generation sequencing platforms often make DNA reads only a few hundred letters long, however they can scan millions of short pieces of DNA at once. Introduction to Next Generation Sequencing (NGS) Andrew Parrish Exeter, 2 nd November 2017 wendy smith school of computing science, The past, present, and future of DNA sequencing - . At the 2015 American Association of Clinical Chemistry Annual Meeting, Dr. Linnea Baudhuin, Co-Director of the Mayo Clinic’s Next-Generation Sequencing Core Laboratory, moderated and co-presented a workshop on next-generation sequencing (NGS). Generations of Sequencing Technologies: From First to Next Generation Mehdi Kchouk1,3*, Jean-François Gibrat2 and Mourad Elloumi3 1Faculty of Sciences of Tunis (FST), Tunis El-Manar University, Tunisia 2Research Unit Applied Mathematics and Computer Science, from Genomes to the Environment' (MaIAGE), Jouy en Josas, France 3Laboratory of Technologies of Information … We'll introduce the Central Dogma of Molecular Biology and cover how next-generation sequencing can be used to measure DNA, RNA, and epigenetic patterns. Next generation sequencing Dayananda Salam. Then the researcher can add the pieces back into the full genome picture using specialized software. Welcome to Next-Generation Sequencing The five years since the introduction of NGS technology have seen a major transformation in the way scientists extract genetic information from biological systems, revealing limitless insight about • random priming of RNA DNA methylation analysis • sequencing bisulfite-converted DNA methylation-sensitive restriction digest enriched fragments Examine chromatin modifications • Quantify in vivo protein-DNA interactions using the combination of chromatin immunoprecipitation and sequencing (ChIP-Seq) Computational Biology Research Group, 454 vs Solexa • Homopolymers (AAAAA..) • Read length: 400 bp • Number of reads: 400.000 • Per-base cost greater • De novo assembly, metagenomics • Read length: 40 bp • Number of reads: millions • Per-base cost cheaper • Ideal for application requiring short reads: ncRNA, Problem: Huge Amount of Image Data • Raw image data huge: 1-2 TB for the Solexa, more for ABI-SOLID, less for 454 • The images are immediately processed into intensity data (spots w/ location and brightness) • Intensity data is then processed into basecalls (A, C, T, or G plus a quality score for each) • Basecall data is on the order of 5-10 GB per run (or a week of runs for 454), Adapted from John McPherson, OICR 2009/10 AB SOLiDv3 120Gb, 100 bp reads 100 Gb Illumina HiSeq 100Gb, 150bp reads 10 Gb 1 Gb 454 GS FLX Titanium bases per machine run 0.4-0.6 Gb, 100-400 bp reads 100 Mb 10 Mb ABI capillary sequencer (0.04-0.08 Mb, 450-800 bp reads 1 Mb 10 bp 100 bp 1,000 bp read length, Storage becoming a real problem Kahn, 2011, Science, Lower Cost = More Innovation • As sequencing becomes cheaper, more investigators can use it for routine assays • Leads to variations and absolutely novel applications • Replacement of other really good technologies, Lower Cost = More samples • More patients in association studies • More replicates in all other assays • More permutations, Bioinformatics is the Bottleneck • Sequencing is a commodity – can easily be outsourced • Bioinformatics is the essential point of the science • Data analysis and discovery of meaning in results • As the data throughput increases, the cost and time spent on analysis increase more than linearly, © 2020 SlideServe | Powered By DigitalOfficePro, - - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -. 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