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• W13383624 abstract "During the past five years, new high-throughput DNA sequencing technologies have emerged; these technologies are collectively referred to as next generation sequencing (NGS). By virtue of sequencing clonally amplified DNA templates or single DNA molecules in a massively parallel fashion in a flow cell, NGS provides both qualitative and quantitative sequence data. This combination of information has made NGS the technology of choice for complex genetic analyses that were previously either technically infeasible or cost prohibitive. As a result, NGS has had a fundamental and broad impact on many facets of biomedical research. In contrast, the dissemination of NGS into the clinical diagnostic realm is in its early stages. Though NGS is powerful and can be envisioned to have multiple applications in clinical diagnostics, the technology is currently complex. Successful adoption of NGS into the clinical laboratory will require expertise in both molecular biology techniques and bioinformatics. The current report presents principles that underlie NGS including sequencing library preparation, sequencing chemistries, and an introduction to NGS data analysis. These concepts are subsequently further illustrated by showing representative results from a case study using NGS for targeted resequencing of genes implicated in hypertrophic cardiomyopathy. During the past five years, new high-throughput DNA sequencing technologies have emerged; these technologies are collectively referred to as next generation sequencing (NGS). By virtue of sequencing clonally amplified DNA templates or single DNA molecules in a massively parallel fashion in a flow cell, NGS provides both qualitative and quantitative sequence data. This combination of information has made NGS the technology of choice for complex genetic analyses that were previously either technically infeasible or cost prohibitive. As a result, NGS has had a fundamental and broad impact on many facets of biomedical research. In contrast, the dissemination of NGS into the clinical diagnostic realm is in its early stages. Though NGS is powerful and can be envisioned to have multiple applications in clinical diagnostics, the technology is currently complex. Successful adoption of NGS into the clinical laboratory will require expertise in both molecular biology techniques and bioinformatics. The current report presents principles that underlie NGS including sequencing library preparation, sequencing chemistries, and an introduction to NGS data analysis. These concepts are subsequently further illustrated by showing representative results from a case study using NGS for targeted resequencing of genes implicated in hypertrophic cardiomyopathy. Next generation sequencing (NGS) refers to high-throughput sequencing technologies that have emerged during the past five years. These technologies share a fundamental process in which clonally amplified DNA templates, or single DNA molecules, are sequenced in a massively parallel fashion in a flow cell.1Mardis ER Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet. 2008; 9: 387-402Crossref PubMed Scopus (1553) Google Scholar2Mardis ER The impact of next-generation sequencing technology on genetics.Trends Genet. 2008; 24: 133-141Abstract Full Text Full Text PDF PubMed Scopus (1559) Google Scholar3Metzker ML Sequencing technologies - the next generation.Nat Rev Genet. 2010; 11: 31-46Crossref PubMed Scopus (4998) Google Scholar Sequencing is conducted in either a stepwise iterative process or in a continuous real-time manner. By virtue of the highly parallel process, each clonal template or single molecule is “individually” sequenced and can be counted among the total sequences generated. The high-throughput combination of qualitative and quantitative sequence information generated has allowed analyses that were previously either not technically possible or cost prohibitive. This has positioned NGS as the method of choice for large-scale complex genetic analyses including whole genome and transcriptome sequencing, metagenomic characterization of microbial species in environmental and clinical samples, elucidation of DNA binding sites for chromatin and regulatory proteins, and targeted resequencing of regions of the human genome identified by linkage analyses and genome wide association studies.4Yeager M Xiao N Hayes RB Bouffard P Desany B Burdett L Orr N Matthews C Qi L Crenshaw A Markovic Z Fredrikson KM Jacobs KB Amundadottir L Jarvie TP Hunter DJ Hoover R Thomas G Harkins TT Chanock SJ Comprehensive resequence analysis of a 136 kb region of human chromosome 8q24 associated with prostate and colon cancers.Hum Genet. 2008; 124: 161-170Crossref PubMed Scopus (88) Google Scholar5Wang C Mitsuya Y Gharizadeh B Ronaghi M Shafer RW Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.Genome Res. 2007; 17: 1195-1201Crossref PubMed Scopus (353) Google Scholar6Urich T Lanzen A Qi J Huson DH Schleper C Schuster SC Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome.PLoS ONE. 2008; 3: e2527Crossref PubMed Scopus (557) Google Scholar7Keijser BJ Zaura E Huse SM van der Vossen JM Schuren FH Montijn RC ten Cate JM Crielaard W Pyrosequencing analysis of the oral microflora of healthy adults.J Dent Res. 2008; 87: 1016-1020Crossref PubMed Scopus (477) Google Scholar8Wang Z Gerstein M Snyder M RNA-Seq: a revolutionary tool for transcriptomics.Nat Rev Genet. 2009; 10: 57-63Crossref PubMed Scopus (8465) Google Scholar9Park PJ ChIP-seq: advantages and challenges of a maturing technology.Nat Rev Genet. 2009; 10: 669-680Crossref PubMed Scopus (1305) Google Scholar10Beck S Rakyan VK The methylome: approaches for global DNA methylation profiling.Trends Genet. 2008; 24: 231-237Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar11Turnbaugh PJ Hamady M Yatsunenko T Cantarel BL Duncan A Ley RE Sogin ML Jones WJ Roe BA Affourtit JP Egholm M Henrissat B Heath AC Knight R Gordon JI A core gut microbiome in obese and lean twins.Nature. 2008; 457: 480-484Crossref PubMed Scopus (5567) Google Scholar12Ding L Getz G Wheeler DA Mardis ER McLellan MD Cibulskis K Sougnez C Greulich H Muzny DM Morgan MB Fulton L Fulton RS Zhang Q Wendl MC Lawrence MS Larson DE Chen K Dooling DJ Sabo A Hawes AC Shen H Jhangiani SN Lewis LR Hall O Zhu Y Mathew T Ren Y Yao J Scherer SE Clerc K Metcalf GA Ng B Milosavljevic A Gonzalez-Garay ML Osborne JR Meyer R Shi X Tang Y Koboldt DC Lin L Abbott R Miner TL Pohl C Fewell G Haipek C Schmidt H Dunford-Shore BH Kraja A Crosby SD Sawyer CS Vickery T Sander S Robinson J Winckler W Baldwin J Chirieac LR Dutt A Fennell T Hanna M Johnson BE Onofrio RC Thomas RK Tonon G Weir BA Zhao X Ziaugra L Zody MC Giordano T Orringer MB Roth JA Spitz MR Wistuba II Ozenberger B Good PJ Chang AC Beer DG Watson MA Ladanyi M Broderick S Yoshizawa A Travis WD Pao W Province MA Weinstock GM Varmus HE Gabriel SB Lander ES Gibbs RA Meyerson M Wilson RK Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2169) Google Scholar While NGS has experienced wide dissemination throughout biomedical research, its translation into molecular diagnostics is just beginning. This report reviews key process steps of NGS, including library preparation, sequencing, and data analysis. Concepts are subsequently illustrated in the context of a diagnostic application the authors are developing for targeted resequencing of multiple genes whose mutational spectrum lead to the overlapping clinical phenotype of hypertrophic cardiomyopathy. NGS technologies share general processing steps, as shown in Figure 1, while differing in specific technical details. A major first step in this process is preparation of a “library” comprising DNA fragments ligated to platform-specific oliognucleotide adapters. The input nucleic acid can be genomic DNA, standard or long-range PCR amplicons, or cDNA. To achieve fragmentation, the input nucleic acid is subjected to shearing by nebulization, sonication, or enzymatic digestion. The goal is to generate random overlapping fragments typically in the size range of 150–600 bp depending on platform and application requirements. Fragmentation by nebulization uses compressed air flowing through an aqueous solution of nucleic acid for several minutes. This approach is prone to volume loss and potential sample cross-contamination. Further, a broad distribution of fragment sizes is generated, which is disadvantageous when a smaller and more restricted size fragment population is needed. Sonication devices for closed tube fragmentation in the $10-$15,000 range are available, including those manufactured by Diagenode (Sparta, NJ) and Misonix (Farmingdale, NY). However, the premiere instrumentation for fragmentation, in our experience, is manufactured by Covaris (Woburn, MA), which uses acoustic wave energy transmitted into a closed tube containing an aqueous DNA solution. This results in formation and collapse of air bubbles, which generate microscale water jets that cause physical shearing of the nucleic acid. Covaris instruments, which cost $45,000-$125,000 depending on sample throughput capacity, generate the most reproducible and tunable fragment size distributions. In addition, New England Biolabs (Ipswich, MA) has recently introduced a promising enzymatic digestion technology, dsDNA Fragmentase, that uses two enzymes, one that randomly nicks dsDNA and the other that recognizes the nicked site and cuts on the opposite strand to produce dsDNA breaks. Regardless of fragmentation method, optimum conditions must be empirically established based on the size of input nucleic acid and the desired fragment size distribution, with “tighter” distributions generally preferred so as to maximize representation of sequences in the library. Fragmented nucleic acids have terminal overhangs, which require blunt end repair and phosphorylation. Commonly, fragments are incubated with Klenow (3′ to 5′ exonuclease minus), T4 DNA polymerase (3′ to 5′ exonuclease plus), and polynucleotide kinase in the presence of dNTPs and ATP. T4 DNA polymerase removes 3′ overhangs and the polymerase activity of Klenow and T4 DNA polymerase fill in 5′ overhangs. Phosphorylation of 5′ ends occurs in parallel via T4 polynucleotide kinase activity. Repaired fragments are purified using a spin column or magnetic beads. In some platform protocols, monoadenylation of 3′ ends is subsequently performed using Klenow and dATP. This enhances the efficiency of ligation to platform specific oligonucleotide adapters (with T overhangs). Ligation products are often size separated by gel electrophoresis, and a specific size range is selected compatible with a given platform or application. The adapter modified fragments constitute the “library” of overlapping sequences. For some protocols, the library concentration needs to be increased and this is accomplished by PCR with primers complementary to adapter sequences. The next major step is to prepare the “library” for massively parallel sequencing. The first wave of NGS platforms manufactured by Roche 454 (Branford, CT), Life Technologies (Carlsbad, CA), and Illumina (San Diego, CA) require their respective libraries to be clonally amplified before sequencing. For the Roche-454 GS-FLX (and GS-Junior) and Life Technologies SOLiD platforms, clonal amplification uses emulsion PCR and requires hybridizing the adapter modified fragment library to beads that display oligonucleotides with sequences complementary to adapter sequences.13McKernan KJ Peckham HE Costa GL McLaughlin SF Fu Y Tsung EF Clouser CR Duncan C Ichikawa JK Lee CC Zhang Z Ranade SS Dimalanta ET Hyland FC Sokolsky TD Zhang L Sheridan A Fu H Hendrickson CL Li B Kotler L Stuart JR Malek JA Manning JM Antipova AA Perez DS Moore MP Hayashibara KC Lyons MR Beaudoin RE Coleman BE Laptewicz MW Sannicandro AE Rhodes MD Gottimukkala RK Yang S Bafna V Bashir A MacBride A Alkan C Kidd JM Eichler EE Reese MG De La Vega FM Blanchard AP Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.Genome Res. 2009; 19: 1527-1541Crossref PubMed Scopus (396) Google Scholar14Wheeler DA Srinivasan M Egholm M Shen Y Chen L McGuire A He W Chen YJ Makhijani V Roth GT Gomes X Tartaro K Niazi F Turcotte CL Irzyk GP Lupski JR Chinault C Song XZ Liu Y Yuan Y Nazareth L Qin X Muzny DM Margulies M Weinstock GM Gibbs RA Rothberg JM The complete genome of an individual by massively parallel DNA sequencing.Nature. 2008; 452: 872-876Crossref PubMed Scopus (1404) Google Scholar15Margulies M Egholm M Altman WE Attiya S Bader JS Bemben LA Berka J Braverman MS Chen YJ Chen Z Dewell SB Du L Fierro JM Gomes XV Godwin BC He W Helgesen S Ho CH Irzyk GP Jando SC Alenquer ML Jarvie TP Jirage KB Kim JB Knight JR Lanza JR Leamon JH Lefkowitz SM Lei M Li J Lohman KL Lu H Makhijani VB McDade KE McKenna MP Myers EW Nickerson E Nobile JR Plant R Puc BP Ronan MT Roth GT Sarkis GJ Simons JF Simpson JW Srinivasan M Tartaro KR Tomasz A Vogt KA Volkmer GA Wang SH Wang Y Weiner MP Yu P Begley RF Rothberg JM Genome sequencing in microfabricated high-density picolitre reactors.Nature. 2005; 437: 376-380Crossref PubMed Scopus (5964) Google Scholar Hybridization is performed under limiting dilution conditions to achieve hybridization of single library fragments to single beads. This is followed by emulsion PCR, wherein single beads containing single library fragments are segregated into water in oil microdroplets and thermocycled. During cycling, tens of thousands to millions of copies of the starting single fragment sequence are produced on the bead surface (with amplification amount platform specific). Post-PCR, the emulsion is disrupted and beads containing clonally amplified templates are enriched and individually deposited into wells (Roche-454 picotiter plate) or onto a surface-modified glass slide flow cell (Life Technologies SOLiD). The deposition of beads needs to be performed under conditions in which only a single bead is deposited per well or, with the SOLiD technology, beads need to be sufficiently spatially separated on the slide surface. The presence of clonally amplified DNA on the bead surface is required to generate sufficient signal for optical capture from each bead during sequencing. The replicate copies are sequenced “in unison” to yield one sequence read per bead. For the Illumina Genome Analyzer platform, adapter modified library fragments are automatically dispensed under limiting dilution conditions onto a glass slide flow cell that displays oligonucleotides complementary to Illumina adapter sequences.16Bentley DR Balasubramanian S Swerdlow HP Smith GP Milton J Brown CG Hall KP Evers DJ Barnes CL Bignell HR Boutell JM Bryant J Carter RJ Keira Cheetham R Cox AJ Ellis DJ Flatbush MR Gormley NA Humphray SJ Irving LJ Karbelashvili MS Kirk SM Li H Liu X Maisinger KS Murray LJ Obradovic B Ost T Parkinson ML Pratt MR Rasolonjatovo IM Reed MT Rigatti R Rodighiero C Ross MT Sabot A Sankar SV Scally A Schroth GP Smith ME Smith VP Spiridou A Torrance PE Tzonev SS Vermaas EH Walter K Wu X Zhang L Alam MD Anastasi C Aniebo IC Bailey DM Bancarz IR Banerjee S Barbour SG Baybayan PA Benoit VA Benson KF Bevis C Black PJ Boodhun A Brennan JS Bridgham JA Brown RC Brown AA Buermann DH Bundu AA Burrows JC Carter NP Castillo N Chiara ECM Chang S Neil Cooley R Crake NR Dada OO Diakoumakos KD Dominguez-Fernandez B Earnshaw DJ Egbujor UC Elmore DW Etchin SS Ewan MR Fedurco M Fraser LJ Fuentes Fajardo KV Scott Furey W George D Gietzen KJ Goddard CP Golda GS Granieri PA Green DE Gustafson DL Hansen NF Harnish K Haudenschild CD Heyer NI Hims MM Ho JT Horgan AM Hoschler K Hurwitz S Ivanov DV Johnson MQ James T Huw Jones TA Kang GD Kerelska TH Kersey AD Khrebtukova I Kindwall AP Kingsbury Z Kokko-Gonzales PI Kumar A Laurent MA Lawley CT Lee SE Lee X Liao AK Loch JA Lok M Luo S Mammen RM Martin JW McCauley PG McNitt P Mehta P Moon KW Mullens JW Newington T Ning Z Ling Ng B Novo SM O'Neill MJ Osborne MA Osnowski A Ostadan O Paraschos LL Pickering L Pike AC Chris Pinkard D Pliskin DP Podhasky J Quijano VJ Raczy C Rae VH Rawlings SR Chiva Rodriguez A Roe PM Rogers J Rogert Bacigalupo MC Romanov N Romieu A Roth RK Rourke NJ Ruediger ST Rusman E Sanches-Kuiper RM Schenker MR Seoane JM Shaw RJ Shiver MK Short SW Sizto NL Sluis JP Smith MA Ernest Sohna Sohna J Spence EJ Stevens K Sutton N Szajkowski L Tregidgo CL Turcatti G Vandevondele S Verhovsky Y Virk SM Wakelin S Walcott GC Wang J Worsley GJ Yan J Yau L Zuerlein M Mullikin JC Hurles ME McCooke NJ West JS Oaks FL Lundberg PL Klenerman D Durbin R Smith AJ Accurate whole human genome sequencing using reversible terminator chemistry.Nature. 2008; 456: 53-59Crossref PubMed Scopus (2520) Google Scholar Surface bound individual fragment molecules are clonally amplified using an isothermal bridge amplification method that generates clonal “clusters” of approximately 1000 identical molecules per cluster. By this approach, one fragment is bound to one surface oligonucleotide, undergoes cluster generation, and the replicate copies are sequenced to yield one sequence read. For the Roche 454, Life Technologies, and Illumina platforms, library preparation is a multistep manual process of pipetting, incubations, and purifications of enzymatic reaction products using spin columns or magnetic beads. For the Illumina technology, a gel purification-based size selection of the library is commonly performed.17Quail MA Kozarewa I Smith F Scally A Stephens PJ Durbin R Swerdlow H Turner DJ A large genome center's improvements to the Illumina sequencing system.Nat Methods. 2008; 5: 1005-1010Crossref PubMed Scopus (567) Google Scholar Clonal amplification by emulsion PCR adds considerable complexity to sample processing. However, new front-end automation devices for the emulsion PCR steps have been developed by Roche 454 and Life Technologies. In addition, Epicenter Biotechnologies (Madison, WI) has recently introduced a novel, less manual approach to library preparation wherein fragmentation and adapter addition are accomplished using in vitro transposition and PCR. A Transposome complex comprising free transposon oligonucleotide ends and a transposase is incubated with input DNA resulting in nearly random fragmentation with sizes controlled by enzyme concentration and incubation time. During fragmentation the transposon associated oligonucleotides are covalently attached to the 5′ ends of the target fragment. The fragments are then amplified using tailed PCR primers in which the 5′ primer region contains either Roche 454 or Illumina library specific adapter sequences and the 3′ region is complementary to the transposon oliognucleotides. The resulting library is then ready for either emulsion PCR or bridge amplification. Important quality control steps are essential during library preparation, notably assuring that fragmentation yielded an appropriate size distribution for the desired application and platform being used. Increasingly, determinations of fragment distributions, size changes due to adapter ligation, and library quantification are performed with high-resolution electrophoresis instruments (eg, Agilent BioAnalyzer, Santa Clara, CA). Sequencing of clonally amplified templates on first wave platforms uses nucleotides or their labeled analogs, or oligonucleotide probes, that are incorporated in a stepwise, iterative manner with incorporation events optically monitored and bioinformatically converted to sequence. Chemistries includes pyrosequencing (Roche 454), sequencing by reversible dye terminators (Illumina), and sequencing by sequential ligation of oligonucleotide probes (Life Technologies SOLiD). For pyrosequencing on the Roche 454 GS-FLX and new GS-Junior platforms, dATP, dCTP, dGTP, or dTTP and polymerase are sequentially flowed over the picotitre plate containing bead bound clonally amplified DNA templates. When incorporation of a complementary nucleotide occurs on a growing strand in an individual well, pyrophosphate is released, which drives luciferase-mediated light generation in the well. The luminescent bursts are optically captured with a high-sensitivity CCD camera. Luminescence intensity is directly proportional to the number of nucleotides incorporated, thus homopolymer signals are greater than single base additions and length-dependent. However, accuracy of homopolymer determination decreases with increasing homopolymer length.15Margulies M Egholm M Altman WE Attiya S Bader JS Bemben LA Berka J Braverman MS Chen YJ Chen Z Dewell SB Du L Fierro JM Gomes XV Godwin BC He W Helgesen S Ho CH Irzyk GP Jando SC Alenquer ML Jarvie TP Jirage KB Kim JB Knight JR Lanza JR Leamon JH Lefkowitz SM Lei M Li J Lohman KL Lu H Makhijani VB McDade KE McKenna MP Myers EW Nickerson E Nobile JR Plant R Puc BP Ronan MT Roth GT Sarkis GJ Simons JF Simpson JW Srinivasan M Tartaro KR Tomasz A Vogt KA Volkmer GA Wang SH Wang Y Weiner MP Yu P Begley RF Rothberg JM Genome sequencing in microfabricated high-density picolitre reactors.Nature. 2005; 437: 376-380Crossref PubMed Scopus (5964) Google Scholar After incorporations events are recorded, residual nucleotides are washed out of the flow cell and the process is repeated. Illumina sequencing uses a mixture of four fluorescently unique reversible dye terminators that are simultaneously introduced into the flow cell, along with DNA polymerase. Incorporation of complementary bases into individual clusters is recorded by virtue of base specific fluorescent emission spectra. The fluor and termination moieties, linked to the nucleotide base and 3′ deoxyribose sugar position, respectively, are then cleaved and washed away. Successive cycles of dye terminator mixture and DNA polymerase introduction, incorporation, and cleavage yield chain elongation. Sequencing by ligation on the SOLiD platform involves the iterative introduction of combinations of fluorescently unique oligonucleotide probes containing specific interrogation nucleotides and degenerate nucleotides. Postannealing and ligation, fluorescence is recorded and the labeled section of each probe is cleaved and washed away. Sequencing by ligation of oligonucleotide probes is conceptually different from other NGS technologies in that sequence is inferred from probe hybridization events and includes the process of each base being interrogated twice for each sequence read generated.13McKernan KJ Peckham HE Costa GL McLaughlin SF Fu Y Tsung EF Clouser CR Duncan C Ichikawa JK Lee CC Zhang Z Ranade SS Dimalanta ET Hyland FC Sokolsky TD Zhang L Sheridan A Fu H Hendrickson CL Li B Kotler L Stuart JR Malek JA Manning JM Antipova AA Perez DS Moore MP Hayashibara KC Lyons MR Beaudoin RE Coleman BE Laptewicz MW Sannicandro AE Rhodes MD Gottimukkala RK Yang S Bafna V Bashir A MacBride A Alkan C Kidd JM Eichler EE Reese MG De La Vega FM Blanchard AP Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.Genome Res. 2009; 19: 1527-1541Crossref PubMed Scopus (396) Google Scholar Resulting read lengths vary between chemistries and have been progressively increasing as each technology evolves. Of the first wave technologies, Roche 454 pyrosequencing read lengths are the longest at 400 plus bases. As of this writing, a new platform, requiring clonally amplified templates, has been announced by Ion Torrent (Guilford, CT), whose CEO Jonathan Rothberg earlier founded 454 Life Sciences. The Ion Torrent platform takes clonally amplified DNA templates and individually sequences them on a semiconductor chip consisting of an array of about 1.55 million 3.5-micrometer wells. Underneath the wells is an ion-sensitive layer and one electronic sensor per well. Unmodified nucleotides are sequentially added in the presence of DNA polymerase. With complementary base incorporation, hydrogen ions are released during formation of the 5′ to 3′ phosphodiester bond. The released hydrogen ions decrease the pH of the solution in the well proportional to the number of bases incorporated and the decrease is registered by the sensing system, which is a microscale solid-state pH meter. This approach, which generates read lengths in the 100 base range, represents the first NGS technology that does not depend on light or fluorescent detection. While the Roche 454, Life Technologies SOLiD, and Illumina platforms currently dominate the field, a second wave of platforms based on single molecule DNA sequencing (SMS) is emerging. Commercially available SMS platforms are those from Helicos BioSciences (Cambridge, MA) and Pacific Biosciences (Menlo Park, CA), with a third platform in development by Life Technologies. Single DNA molecule sequencing inherently reduces the complexity of library preparation in that clonal amplification before sequencing is eliminated. The Helicos BioSciences HeliScope has a library preparative procedure in which DNA fragments are enzymatically polyA 3′ tailed, purified, and hybridized to oligo dT attached to a glass slide flow cell surface. For sequencing on the Heliscope, a variation of dye terminator chemistry comprising one color Cy5 fluorescently labeled, 3′ unblocked reversible dye terminators is used.18Milos PM Emergence of single-molecule sequencing and potential for molecular diagnostic applications.Expert Rev Mol Diagn. 2009; 9: 659-666Crossref PubMed Scopus (25) Google Scholar19Ozsolak F Platt AR Jones DR Reifenberger JG Sass LE McInerney P Thompson JF Bowers J Jarosz M Milos PM Direct RNA sequencing.Nature. 2009; 461: 814-818Crossref PubMed Scopus (336) Google Scholar20Goren A Ozsolak F Shoresh N Ku M Adli M Hart C Gymrek M Zuk O Regev A Milos PM Bernstein BE Chromatin profiling by directly sequencing small quantities of immunoprecipitated DNA.Nat Methods. 2010; 7: 47-49Crossref PubMed Scopus (95) Google Scholar21Harris TD Buzby PR Babcock H Beer E Bowers J Braslavsky I Causey M Colonell J Dimeo J Efcavitch JW Giladi E Gill J Healy J Jarosz M Lapen D Moulton K Quake SR Steinmann K Thayer E Tyurina A Ward R Weiss H Xie Z Single-molecule DNA sequencing of a viral genome.Science. 2008; 320: 106-109Crossref PubMed Scopus (522) Google Scholar In this approach, sequencing is conducted in a stepwise manner, with each incorporation event monitored at the single molecule level using total internal reflection fluorescence microscopy. The Pacific BioSciences SMS technology represents the first “real-time” sequencing method based on continuous monitoring of DNA polymerase-mediated incorporation of labeled nucleotide analogues.22Korlach J Bibillo A Wegener J Peluso P Pham TT Park I Clark S Otto GA Turner SW Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides.Nucleosides Nucleotides Nucleic Acids. 2008; 27: 1072-1083Crossref PubMed Scopus (65) Google Scholar23Korlach J Marks PJ Cicero RL Gray JJ Murphy DL Roitman DB Pham TT Otto GA Foquet M Turner SW Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures.Proc Natl Acad Sci USA. 2008; 105: 1176-1181Crossref PubMed Scopus (180) Google Scholar24Eid J Fehr A Gray J Luong K Lyle J Otto G Peluso P Rank D Baybayan P Bettman B Bibillo A Bjornson K Chaudhuri B Christians F Cicero R Clark S Dalal R Dewinter A Dixon J Foquet M Gaertner A Hardenbol P Heiner C Hester K Holden D Kearns G Kong X Kuse R Lacroix Y Lin S Lundquist P Ma C Marks P Maxham M Murphy D Park I Pham T Phillips M Roy J Sebra R Shen G Sorenson J Tomaney A Travers K Trulson M Vieceli J Wegener J Wu D Yang A Zaccarin D Zhao P Zhong F Korlach J Turner S Real-time DNA sequencing from single polymerase molecules.Science. 2008; 323: 133-138Crossref PubMed Scopus (2434) Google Scholar For library preparation, fragmented DNA is end repaired, monoadenylated, and ligated to adapters that form a stem loop structure on each fragment end. Molecular complexes are then formed in solution comprised of individual DNA library fragments with stem loop adapters that are primed with a sequencing primer complementary to adapter loop sequences, and phi 29 DNA polymerase. Under limiting dilution conditions, individual complexes are deposited into nanoscale wells present in a highly parallel flow cell configuration. Each nanoscale well is optically monitored by a zero mode waveguide. To immobilize the complex, the polymerase is biotinylated and bound to streptavidin on the well floor so as to be optimally oriented in the zero mode waveguide. To initiate DNA polymerization and hence sequencing, divalent cation and four differently labeled nucleotide analogs are added. A new class of labeled nucleotide analogs was developed for this technology wherein a hexaphosphate moiety is linked to the 5′ position of the deoxyribose sugar. Through a phosphoester bond, nucleotide specific fluors are coupled to the terminal phosphate. As a complementary phospholabeled nucleotide is incorporated into the growing DNA strand, its respective emission fluorescence is observed through excitation in the zero mode waveguide. With incorporation, the phospholinked fluorescent moiety is cleaved and rapidly diffuses away. Successive incorporation events take place at a rate of 2 to 4 bases per second. The ability to distinguish specific incorporation signals from background, due to noncomplementary nucleotide sampling by the polymerase and random nucleotide diffusion, is based on the longer time that a complementary base is “held” by the polymerase. Each incorporation event generates a pulse of nucleotide specific fluorescent emission followed by a return to baseline background fluorescence before the next incorporation event. The phi29 DNA polymerase is a hig" @default.
• W13383624 created "2016-06-24" @default.
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• W13383624 date "2010-09-01" @default.
• W13383624 modified "2023-10-16" @default.
• W13383624 title "Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy" @default.
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