review article next generation sequencing: potential and...

Review ArticleNext Generation Sequencing Potential and Application in DrugDiscovery

Navneet Kumar Yadav1 Pooja Shukla12 Ankur Omer12 Shruti Pareek3 and R K Singh12

1 Hematological Facility Division of Toxicology CSIR-Central Drug Research Institute BS-101 Sector 10 Jankipuram ExtensionSitapur Road PO Box 173 Lucknow 226031 India

2 Academy of Scientific and Innovative Research New Delhi 110 001 India3 Jaipur National University Jaipur Rajasthan 302017 India

Correspondence should be addressed to R K Singh rktoxigmailcom

Received 12 August 2013 Accepted 18 October 2013 Published 5 February 2014

Academic Editors Z P Cakar and S Rodrıguez-Couto

Copyright copy 2014 Navneet Kumar Yadav et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The world has now entered into a new era of genomics because of the continued advancements in the next generationhigh throughput sequencing technologies which includes sequencing by synthesis-fluorescent in situ sequencing (FISSEQ)pyrosequencing sequencing by ligation using polony amplification supported oligonucleotide detection (SOLiD) sequencing byhybridization along with sequencing by ligation and nanopore technology Great impacts of these methods can be seen for solvingthe genome related problems of plant and animal kingdom that will open the door of a new era of genomics This may ultimatelyovercome the Sanger sequencing that ruled for 30 years NGS is expected to advance and make the drug discovery process morerapid

1 Introduction

In 1977 Fred Sanger et al published two methodology basedpapers on the rapid determination of DNA sequences thathelped to transform biology and provided a new tool fordeciphering complete genes and later the entire genome [1 2]The methods dramatically improved existing DNA sequenc-ing techniques developed by Maxam and Gilbert publishedin the same year and Sanger and Coulsonrsquos own ldquoplus andminusrdquomethod published 2 years earlier [3 4]The advantageof dealing with less toxic chemicals and radioisotopes madeldquoSanger sequencingrdquo the only DNA sequencing method usedfor the next 30 yearsThegel based sequencing technology hasundergone dramatic improvement in throughput from oneparallelization automation and refinement of sequencingmethods and chemistry Recent advances in microfibrationhave resulted in further improvements of Sanger sequencingby multiplexing and miniaturization These advances havebeen reviewed by Metzker [5] Despite many improvements

the gel based Sanger sequencing technology still faces draw-backs in the name of cost and low throughput For achievinghigh throughput many commercial companies and scientificlabs have come up with different ways of high throughputsequencing with a reasonable cost

The technologies named together as next generationsequencing technologies include sequencing by synthesisdeveloped by 454 Life-Science [6] sequencing by liga-tion sequencing by hybridization single molecule DNAsequencing nanopore sequencing and multiplex polonysequencing of George Churchrsquos lab [7] Next generationsequencing has revolutionary impacts on genetic applicationslike metagenomics comparative genomics high throughputpolymorphism detection analysis of small RNAs mutationscreening transcriptome profiling methylation profilingand chromatin remodeling The major measurements for thesuccess of the next generation technology are sequence (readlength) sequence quality high throughput and low cost

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 802437 7 pageshttpdxdoiorg1011552014802437

2 The Scientific World Journal

11 Sequencing by Synthesis (SBS) SBS using fluorophore-labeled reversible-terminator nucleotides is the most com-mon platform of sequencing by synthesis It is some-times named ldquofluorescent in situ sequencingrdquo (FISSEQ)The pyrosequencing technology is another SBS technologydeveloped by Ronaghi et al at Stanford University [8 9]It is based on the detection of pyrophosphate (PPi) releasedduring DNA synthesis when inorganic PPi is released afternucleotide incorporation by DNA polymerase The releasedPPi is then converted to ATP by ATP sulfurylase A luciferasereporter enzyme uses the ATP to generate light which isthen detected by a charged couple device (CCD) cameraPyrosequencing has evolved into an ultrahigh throughputsequencing technology with the combination of severaltechnologies such as template carryingmicrobeads depositedin microfabricated picoliter-sized reaction wells connectingto optical fibers [10] 454 Life SciencesRoche Diagnostichas Genome Sequencer 20 System and Genome SequencerFLX System two high throughput commercial sequencingplatforms and DNA helices are fractionated into 300ndash500 bpfragments and linkers are added to their 31015840 and 51015840 endsSingle stranded DNA is isolated and captured on beads Thebeads with DNAs are then emulsified in a ldquowater-in-oilrdquomixture with amplification reagents to create micro reactorsfor Emulsion PCR (emPCR) Finally beads with amplifiedDNAs are loaded onto a picotitre plate for sequencing Theprocedure is given in Figure 1

12 Polony Sequencing George Churchrsquos Laboratory at Har-vard University has developed a high throughput technologybased on polony amplification and FISSEQ [11] Polonyamplification is a method to amplify DNA in situ on a thinpolyacrylamide film [12] The DNA movement is limited inthe polyacrylamide gel so the amplified DNAs are local-ized in the gel and form the so-called ldquopoloniesrdquo that ispolymerase colonies (Figure 1) Up to 5 million polonies(ie 5 million PCRs) can be formed on a single glassmicroscope slideThis sequencing technology has been testedin a bacterial genome and the sequence read lengthwas foundto be about 13 bases per colony [13] Kim et al developeda technology named Polony Multiplex Analysis of GeneExpression (PMAGE) which combines polony amplificationand a sequence-by-ligation method to sequence 14-base tags[14] Up to 5 million polonies can be sequenced in parallel

13 Single Molecule DNA Sequencing Most current sequenc-ing technologies are based on sequencing many identicalcopies of DNA molecules (often amplified) However thereare certain problems associated with sequencing amplifiedmultiple copies of identical DNAs such as achieving thesynchronous priming of each copy of the multiple DNA bythe sequencing primers [15] One way to solve such problemsis to perform sequencing by synthesis method [16] DNA canbe attached to solid support to form single molecule arraysand the single DNA molecule is then sequenced directlyBuzby at Helicos Bio-Science Corp also invented a methodof stabilizing a nucleic acid duplex on a surface for singlemolecule sequencing [17] AppleraCorp invented fluorescent

intercalators that are employed as a donor in fluorescenceresonance energy transfer (FRET) for use in single moleculesequencing reactions [18]

14 Nanopore Sequencing Nanopores are nanometer-scalepores and are regarded as one of the most promising tech-nologies in achieving true real time ultrafast true singlemolecule DNA sequencing [19] Nanopore has been usedfor the detection counting and characterization of singlemolecules by observing the changes in ionic current innanopores when molecules traverse through a nanopore[20 21] The fabrication of nanopores such as the alpha-hemolysin pore and synthetic nanopore has been reviewedrecently [22] Recent progress has made a big step towardultrafast sequencing using nanopore technologies Lagerqvistet al proposed a novel idea to measure the electric currentperpendicular to theDNAbackbone [23] Zhao et al reportedthat a single nucleotide polymorphism can be detected by achange in the threshold voltage of a nanopore [24]

15 Sequencing byHybridization (SBH) SBHwas given in thelate 80s [25] It is amethod of reconstructing aDNA sequencebased on its k-mer content All possible k-nucleotidesoligomers (k-mers) are hybridized to identify overlappingk-mers in an unknown DNA sample These overlapping k-mers are subsequently aligned by algorithms to produce theDNA sequence Drmanac et al reviewed the technology ofsequencing by hybridization [26] In spite of having someproblems using SBHwith predefined probe sets derived froma known sequence has been used to resequence a specificregion of genomic DNA or cDNA for the identification ofsmall deletions insertions polymorphisms and mutations[27]

16 Sequencing by Ligation The first high throughputsequencing by ligation was probably realized by Lynx Ther-apeutics Inc in their early technology of Massively ParallelSignature Sequencing [28] Churchrsquos laboratory at HarvardMedical School advanced the sequencing by ligation methodfor ultra fast and high throughput sequencing [13 14] Theyconverted an epifluorescence for rapid DNA sequencingDNA molecules were amplified in parallel onto micro beadsby emulsion polymerase chain reaction (Figure 1) Millionsof beads were immobilized in a polyacrylamide gel andsequenced using sequencing by ligation method AppliedBiosystem Inc acquired Agencourt Personal Genomics whodeveloped the Supported Oligo Ligation Detection system(SOLiD) for high throughput DNA sequencing

17 Bioinformatics Tools for Analyzing NGS Data Sequencereads generated from NGS technologies are shorter thantraditional Sanger sequences which makes assembly andanalysis of NGS data challenging Some important bioinfor-matics tools are available which is helpful in analyzing NGSdata (Table 1)

18 Applications of NGS Techniques NGS technologies havealready been used for various applications ranging from

The Scientific World Journal 3

TGG

TGG

Enlarged viewSheering to smallfragments

Primer A Primer BDNA to be sequencedPour acrylamide gel with DNAand PCR reagent

In gel PCRamplification

Beforeamplification

SingleDNAmolecule

Polony PCRcolony

Afteramplification

Images of polonies

FISSEQ

Extend withfluorescentdATP scandATP scan

Remove fluorescenceextend with fluorescentdCTP scan

Continue cycles

GCC

GCC

GCC

A

TGG

AC

C

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

(a)

Probes

Matching probe bindsto genomic DNA

DNA ligase bindsmatching probe to

anchor

Each time the

attaches it canbe traced

nucleotide

G

A

T

N N N NN N N N

N N N NN N N N

N N N NN N N N

N N N NN N N N

C

(b)

Extendedbases

Cleavablefluorescent dye

New base being

incorporated

Cleavable cap

ChIP

Extend

Cleave

Measure offluorescence

(c)

Figure 1 Different techniques of next generation sequencing (a) polony sequencing (b) sequencing by Ligation (c) sequencing by synthesis


Table 1 Important bioinformatics tools for analysis of NGS data

Alignment assembly and visualization toolsVelvet (httpwwwebiacuksimzerbinovelvet) tool for de novo assembly of short and paired readsEULER (httpngslibi-medacatnode64) tool to generate short-read assembly and facilitate assembly of combined reads of NGS andSanger sequencingGMAP (httpwwwgenecomsharegmap) program to map and align cDNA sequences to genome sequence using minimal time andmemory and facilitates batch processingSequence variant discovery toolsSNPsniffer tool for SNP discovery specifically designed for Roche454 sequencesSeqMap (httpwww-personalumichedusimjianghuiseqmap) tool to map short sequences to a reference genome and detect multiplesubstitutions and indelsIntegrated toolsNextGENe (httpwwwsoftgeneticscomNextGENehtml) software to analyze NGS data for de novo assembly SNP and indel detectionand transcriptome analysisSeqMan genome analyzer (httpwwwdnastarcomproductsSMGAphp) software with capacity to align NGS and Sanger data anddetect SNPs and also facilitates visualizationCLCbio Genomics Workbench (httpwwwclcbiocom) tool for de novo and reference assembly of Sanger and NGS sequence data SNPdetection and browsingSource httpwikiseqanswerscom

Gene to target

Target to SoC

SoC to candidate

FTIH to POC

POC to phase III

Phase III

Phase IV

PM Vaccine quality control

Drug resistance

DiagnosticsPatient

stratification

Drug rescueRelease

Tran

scrip

tom

ics Gene

variation

Dise

ase

unde

rsta

ndin

g

Epigenetics

Protein interaction

Ab development

Mouse mutants

Encoded library

technology

Figure 2 Application of Next Generation Sequencing in drug discovery SoC standard of care POC proof of concept FTIH first time inhuman

whole genome sequencing resequencing single nucleotidepolymorphism (SNP) structural variation discovery mRNAand noncoding RNA profiling and protein-nucleic acidinteraction assays NGS technologies are becoming a poten-tial tool for gene expression analysis especially for thosespecies having reference genome sequences already available(Figure 2) An overview of NGS applications that are relevantto drug discovery is discussed here

19 Single Nucleotide Polymorphisms (SNP) Discovery SNPsare important genomic resources which can be used in avariety of analyses including physical characteristics likeheight and appearance as well as less obvious traits suchas personality behavior and disease susceptibility Sequence

data generated for parental genotypes of the mapping popu-lation by using NGS technologies can be used for mining theSNPs at large scale [29] SNPs can also significantly influenceresponses to pharmacotherapy and predicting whether thedrugswill produce adverse reactions or notThe developmentof new drugs can be made far cheaper and more rapid byselecting participants in drug trials based on their geneticallydetermined response to the drugs [30]

110 Messenger RNA and Noncoding RNA Profiling Apartfrom SNP discovery expressed regions of a genome canbe detected using NGS technologies The next generationsequencing platforms are capable of identifying expressionlevels of nearly all genes including those rare and species


specific transcripts A similar approach can be applied to largegenomes RNA-seq data can be used to characterize exon-exon splicing events namely cases of alternative splicing [31ndash33]

Noncoding RNA like microRNA (miRNA) is a broadclass of regulatory RNA molecules

The NGS technologies are useful for discovery of non-coding RNA for their short lengths [34] Most studies to datehave used 454 technology because of its early availability todiscover new and different noncoding RNA classes in severalspecies like Chlamydomonas Drosophila Arabidopsis and soforth [35ndash37] A comprehensive study of miRNA in acutemyeloid leukemiawas performedusingNGSwith novel find-ings of differentially expressed miRNAs [38] Transcriptomesequencing has been found to be a powerful tool for detectingnovel gene fusions in cancer cell lines and tissues [39]

111 De Novo Sequencing and Resequencing Metagenomics isdefined as the application of genomics techniques to directlystudy the communities of microbial organisms without iso-lation and cultivation of individual species [40] It involvesthe characterization of the genomes in these communitiesas well as their mRNA protein and metabolic productsThe next generation sequencing technologies have enabledto move metagenomics from a single organism type inisolation to the studies of whole communities NGS enablesthe researchers to avoid the cloning formation and culturesteps which are the major drawbacks of genomics NGSstrategies are straightforward in the following (1) deepsequencing of DNA fragments is conducted on an unculturedsample and (2) short reads are compared against databaseof known sequences using bioinformatics tool like MEGAN[41] with or without assembly and (3) these data are thenused to compute and explore their contents to infer relativeabundances Therefore NGS technologies can be a potenttool for discovery of microorganisms and pathogens

112 Regulatory Protein Binding At low throughput chro-matin immunoprecipitation (ChIP) has enabled regulatoryDNA-protein binding interactions to be elucidated [41] Itis a lengthy process like association of specific antibodyto DNA-binding protein followed by another protein-DNAcross linking agent so that any protein in close associationwith DNA becomes linked Then the cells are lysed DNA isfragmented and the specific antibody is used to precipitatethe protein of interest along with any associated DNAfragment These DNA pieces are subsequently released byreversing the cross linking and identified by southern blottingor qPCR [42] A probe is used to infer the DNA-bindingsite sequence of the protein under study So NGS helps inidentifying the regulatory protein binding site for havingshorter reads and specific binding site and it also provideshigher resolution [43]

113 Exploring Chromatin Packaging Chromatin packagingdenotes the packing of DNA in histones This packaging

determines the transcription of a particular gene Under-standing of this DNA packing that is chromatin packagingis of a great interest An initial 454-based study of genomicDNA packaging into nucleosomes was described for the Celegans by sequencing the DNA isolated from nucleosomecores aftermicrococcal nuclease digestion andmapping themto the reference genome sequence [44] Mikkelsen et alused the Illumina platform to demonstrate the connectionbetween chromatin packaging and gene expression in severaldifferent cell types Mikkelsen et al found that changes inchromatin state at specific promoters reflect changes in geneexpression for the genes they control A better understandingof the chromatin packagingwill provide new strategies for thedevelopment of novel drugs [45]

NGS can also have a central role in the discovery of newgenomic biomarkers detection of mutations personalizedmedicine and pharmacogenetics target identification andvalidation clinical diagnostics vaccine development inves-tigating drug resistance and many others

2 Conclusion

Because of their cost-effectiveness in comparison to theSanger sequencing method and with a wide range of usesNGS approaches have emerged as the dominant genomicstechnology Perhaps most significantly these new sequencershave provided genome-scale sequencing capacity to individ-ual laboratories in addition to larger genome centers Com-pared to Sanger sequencing advantages of the next genera-tion technologies mentioned thus far including 454RocheIlluminaSolexa and ABISOLiD alleviate the need for invivo cloning by clonal amplification of spatially separatedsingle molecules using either emulsion PCR (454Rocheand ABISOLiD) or bridge amplification on solid surface(IlluminaSolexa) We are at the verge of a new genomicrevolution with recent advances in next generation sequenc-ing technologies We will see enormous impacts of thesenext generation sequencingmethods in dealingwith complexbiological problems for example the identification of allsequence changes in drug resistant HIV and drug resistanttuberculosis bacteria (TB) in the delineation of sequencechanges for individual cells during cancer initiation andprogression and in global transcription factormapping ChIPto sequencing [46] In addition NGS technology will createa tremendous impact in medical science It will make routinesequencing of a personrsquos genome sequence of an individualhuman and the initiation of the personal Human GenomeProject [47]

Abbreviations

SBS Sequencing by synthesisFISSEQ Fluorescent in situ sequencingCCD Charged couple deviceemPCR Emulsion PCRPMAGE Polony multiplex analysis of gene expressionFRET Fluorescence resonance energy transfer


SBH Sequencing by hybridizationk-mers k-nucleotides oligomersSOLiD Supported oligo ligation detectionSNP Single nucleotide polymorphismChIP Chromatin immunoprecipitation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Navneet Kumar Yadav Ankur Omer and Pooja Shuklaacknowledge financial support from UGC and DST NewDelhi This is CDRI paper no 742012RKS

References

[1] F Sanger G M Air B G Barrell et al ldquoNucleotide sequenceof bacteriophage 120593X174 DNArdquo Nature vol 265 no 5596 pp687ndash695 1977

[2] F Sanger S Nicklen and A R Coulson ldquoDNA sequencingwith chain-terminating inhibitorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 74 no12 pp 5463ndash5467 1977

[3] A M Maxam and W A Gilbert ldquoNew method for sequencingDNArdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 74 no 2 pp 560ndash564 1977

[4] F Sanger and A R Coulson ldquoNucleotide and amino acidsequences of Gene G of 120593X174rdquo Journal of Molecular Biologyvol 94 pp 441ndash448 1975

[5] M L Metzker ldquoEmerging technologies in DNA sequencingrdquoGenome Research vol 15 no 12 pp 1767ndash1776 2005

[6] M Margulies M Egholm E William et al ldquoGenome sequenc-ing in microfabricated high-density picolitre reactorsrdquo Naturevol 437 pp 376ndash380 2005

[7] J J Shendure G J Porreca N B Reppas et al ldquoMolecularbiology accurate multiplex polony sequencing of an evolvedbacterial genomerdquo Science vol 309 no 5741 pp 1728ndash17322005

[8] M Ronaghi S Karamohamed B Pettersson M Uhlen andP Nyren ldquoReal-time DNA sequencing using detection ofpyrophosphate releaserdquo Analytical Biochemistry vol 242 no 1pp 84ndash89 1996

[9] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[10] J Shendure R D Mitra C Varma and G M ChurchldquoAdvanced sequencing technologies methods and goalsrdquoNature Reviews Genetics vol 5 no 5 pp 335ndash344 2004

[11] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[12] R D Mitra and G M Church ldquoIn situ localized amplificationand contact replication of many individual DNA moleculesrdquoNucleic Acids Research vol 27 no 24 article e34 1999

[13] S Myllykangas J Buenrostro and H P Ji ldquoOverview ofsequencing technology platformsrdquo Bioinformatics For HighThroughput Sequencing pp 11ndash25 2012

[14] J B Kim G J Porreca L Song et al ldquoPolony multiplexanalysis of gene expression (PMAGE) in mouse hypertrophiccardiomyopathyrdquo Science vol 316 no 5830 pp 1481ndash1484 2007

[15] B Lin J Wang and Y Cheng ldquoRecent patents and advances inthe next-generation sequencing technologiesrdquoRecent Patents onBiomedical Engineering vol 1 pp 60ndash67 2008

[16] S N Lapidus P R Buzby and T Harris US20077169560 2007[17] P R Buzby US20077220549 2007[18] H Sun US20070202521A1 2007[19] M Rhee and M A Burns ldquoNanopore sequencing technology

research trends and applicationsrdquo Trends in Biotechnology vol24 no 12 pp 580ndash586 2006

[20] A Meller and D Branton ldquoSingle molecule measurements ofDNA transport through a nanoporerdquoElectrophoresis vol 23 pp2583ndash2591 2002

[21] AMeller LNivon E Brandin J Golovchenko andD BrantonldquoRapid nanopore discrimination between single polynucleotidemoleculesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 97 no 3 pp 1079ndash1084 2000

[22] M Rhee and M A Burns ldquoNanopore sequencing technologynanopore preparationsrdquo Trends in Biotechnology vol 25 no 4pp 174ndash181 2007

[23] J Lagerqvist M Zwolak andM di Ventra ldquoFast DNA sequenc-ing via transverse electronic transportrdquoNano Letters vol 6 no4 pp 779ndash782 2006

[24] Q Zhao G Sigalov V Dimitrov et al ldquoDetecting SNPs using asynthetic nanoporerdquo Nano Letters vol 7 no 6 pp 1680ndash16852007

[25] D Radoje L Ivan B Ivan and C Radomir ldquoSequencing ofmegabase plus DNA by hybridization theory of the methodrdquoGenomics vol 4 pp 114ndash128 1989

[26] R Drmanac S Drmanac G Chui et al ldquoSequencing byhybridization (SBH) advantages achievements and opportu-nitiesrdquo Advances in Biochemical EngineeringBiotechnology vol77 pp 75ndash101 2002

[27] A Schirinzi S Drmanac B Dallapiccola et al ldquoCombinatorialsequencing-by-hybridization analysis of theNF1 generdquoGeneticTesting vol 10 no 1 pp 8ndash17 2006

[28] S Brenner S R Williams E H Vermaas et al ldquoIn vitrocloning of complex mixtures of DNA on microbeads physicalseparation of differentially expressed cDNAsrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol97 no 4 pp 1665ndash1670 2000

[29] V Thakur and R Varshney ldquoChallenges and strategies for nextgeneration sequencing (NGS) data ARrdquo Journal of ComputerScience and Systems Biology vol 3 pp 40ndash42 2010

[30] J Voisey andC PMorris ldquoSNP technologies for drug discoverya current reviewrdquo Current Drug Discovery Technologies vol 5no 3 pp 230ndash235 2008

[31] M Samuel B T Wilhelm and J Bahler ldquoNext-generationsequencing applications beyondGenomesrdquoBiochemical SocietyTransactions vol 36 pp 1091ndash1096 2008

[32] U Nagalakshmi Z Wang K Waern et al ldquoThe transcriptionallandscape of the yeast genome defined by RNA sequencingrdquoScience vol 320 no 5881 pp 1344ndash1349 2008

[33] M Sultan M H Schulz H Richard et al ldquoA global view ofgene activity and alternative splicing by deep sequencing of thehuman transcriptomerdquo Science vol 321 no 5891 pp 956ndash9602008


[34] M Kiriakidou P T Nelson A Kouranov et al ldquoA com-bined computational-experimental approach predicts humanmicroRNA targetsrdquo Genes and Development vol 18 no 10 pp1165ndash1178 2004

[35] J Brennecke A A Aravin A Stark et al ldquoDiscrete small RNA-generating loci as master regulators of transposon activity inDrosophilardquo Cell vol 128 no 6 pp 1089ndash1103 2007

[36] S Alexander K Pouya P Leopold et al ldquoSystematic discoveryand characterization of fly microRNAs using 12 Drosophilagenomesrdquo Genome Research vol 17 no 12 pp 1865ndash1879 2007

[37] K D Kasschau N Fahlgren E J Chapman et al ldquoGenome-wide profiling and analysis of Arabidopsis siRNAsrdquo PLoS Biol-ogy vol 5 no 3 article 57 2007

[38] G Ramsingh D C Koboldt M Trissal et al ldquoCompletecharacterization of the microRNAome in a patient with acutemyeloid leukemiardquo Blood vol 116 no 24 pp 5316ndash5326 2010

[39] C A Maher C Kumar-Sinha X Cao et al ldquoTranscriptomesequencing to detect gene fusions in cancerrdquo Nature vol 458no 7234 pp 97ndash101 2009

[40] K Chen and L Pachter ldquoBioinformatics for whole-genomeshotgun sequencing of microbial communitiesrdquo PLoS Compu-tational Biology vol 1 no 2 article e24 2005

[41] D H Huson A F Auch J Qi and S C Schuster ldquoMEGANanalysis of metagenomic datardquo Genome Research vol 17 no 3pp 377ndash386 2007

[42] M J Solomon P L Larsen and A Varshavsky ldquoMappingprotein-DNA interactions in vivo with formaldehyde evidencethat histone H4 is retained on a highly transcribed generdquo Cellvol 53 no 6 pp 937ndash947 1988

[43] E R Mardis ldquoThe impact of next-generation sequencingtechnology on geneticsrdquo Trends in Genetics vol 24 no 3 pp133ndash141 2008

[44] S M Johnson F J Tan H L McCullough D P Riordanand A Z Fire ldquoFlexibility and constraint in the nucleosomecore landscape of Caenorhabditis elegans chromatinrdquo GenomeResearch vol 16 no 12 pp 1505ndash1516 2006

[45] T S Mikkelsen M Ku D B Jaffe et al ldquoGenome-wide mapsof chromatin state in pluripotent and lineage-committed cellsrdquoNature vol 448 no 7153 pp 553ndash560 2007

[46] D S Johnson A Mortazavi R M Myers and B WoldldquoGenome-wide mapping of in vivo protein-DNA interactionsrdquoScience vol 316 no 5830 pp 1497ndash1502 2007

[47] S Levy G Sutton P C Ng et al ldquoThe diploid genome sequenceof an individual humanrdquoPLoSBiology vol 5 no 10 article e2542007

Submit your manuscripts athttpwwwhindawicom

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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


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Hindawi Publishing Corporationhttpwwwhindawicom

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


11 Sequencing by Synthesis (SBS) SBS using fluorophore-labeled reversible-terminator nucleotides is the most com-mon platform of sequencing by synthesis It is some-times named ldquofluorescent in situ sequencingrdquo (FISSEQ)The pyrosequencing technology is another SBS technologydeveloped by Ronaghi et al at Stanford University [8 9]It is based on the detection of pyrophosphate (PPi) releasedduring DNA synthesis when inorganic PPi is released afternucleotide incorporation by DNA polymerase The releasedPPi is then converted to ATP by ATP sulfurylase A luciferasereporter enzyme uses the ATP to generate light which isthen detected by a charged couple device (CCD) cameraPyrosequencing has evolved into an ultrahigh throughputsequencing technology with the combination of severaltechnologies such as template carryingmicrobeads depositedin microfabricated picoliter-sized reaction wells connectingto optical fibers [10] 454 Life SciencesRoche Diagnostichas Genome Sequencer 20 System and Genome SequencerFLX System two high throughput commercial sequencingplatforms and DNA helices are fractionated into 300ndash500 bpfragments and linkers are added to their 31015840 and 51015840 endsSingle stranded DNA is isolated and captured on beads Thebeads with DNAs are then emulsified in a ldquowater-in-oilrdquomixture with amplification reagents to create micro reactorsfor Emulsion PCR (emPCR) Finally beads with amplifiedDNAs are loaded onto a picotitre plate for sequencing Theprocedure is given in Figure 1

12 Polony Sequencing George Churchrsquos Laboratory at Har-vard University has developed a high throughput technologybased on polony amplification and FISSEQ [11] Polonyamplification is a method to amplify DNA in situ on a thinpolyacrylamide film [12] The DNA movement is limited inthe polyacrylamide gel so the amplified DNAs are local-ized in the gel and form the so-called ldquopoloniesrdquo that ispolymerase colonies (Figure 1) Up to 5 million polonies(ie 5 million PCRs) can be formed on a single glassmicroscope slideThis sequencing technology has been testedin a bacterial genome and the sequence read lengthwas foundto be about 13 bases per colony [13] Kim et al developeda technology named Polony Multiplex Analysis of GeneExpression (PMAGE) which combines polony amplificationand a sequence-by-ligation method to sequence 14-base tags[14] Up to 5 million polonies can be sequenced in parallel

13 Single Molecule DNA Sequencing Most current sequenc-ing technologies are based on sequencing many identicalcopies of DNA molecules (often amplified) However thereare certain problems associated with sequencing amplifiedmultiple copies of identical DNAs such as achieving thesynchronous priming of each copy of the multiple DNA bythe sequencing primers [15] One way to solve such problemsis to perform sequencing by synthesis method [16] DNA canbe attached to solid support to form single molecule arraysand the single DNA molecule is then sequenced directlyBuzby at Helicos Bio-Science Corp also invented a methodof stabilizing a nucleic acid duplex on a surface for singlemolecule sequencing [17] AppleraCorp invented fluorescent

intercalators that are employed as a donor in fluorescenceresonance energy transfer (FRET) for use in single moleculesequencing reactions [18]

14 Nanopore Sequencing Nanopores are nanometer-scalepores and are regarded as one of the most promising tech-nologies in achieving true real time ultrafast true singlemolecule DNA sequencing [19] Nanopore has been usedfor the detection counting and characterization of singlemolecules by observing the changes in ionic current innanopores when molecules traverse through a nanopore[20 21] The fabrication of nanopores such as the alpha-hemolysin pore and synthetic nanopore has been reviewedrecently [22] Recent progress has made a big step towardultrafast sequencing using nanopore technologies Lagerqvistet al proposed a novel idea to measure the electric currentperpendicular to theDNAbackbone [23] Zhao et al reportedthat a single nucleotide polymorphism can be detected by achange in the threshold voltage of a nanopore [24]

15 Sequencing byHybridization (SBH) SBHwas given in thelate 80s [25] It is amethod of reconstructing aDNA sequencebased on its k-mer content All possible k-nucleotidesoligomers (k-mers) are hybridized to identify overlappingk-mers in an unknown DNA sample These overlapping k-mers are subsequently aligned by algorithms to produce theDNA sequence Drmanac et al reviewed the technology ofsequencing by hybridization [26] In spite of having someproblems using SBHwith predefined probe sets derived froma known sequence has been used to resequence a specificregion of genomic DNA or cDNA for the identification ofsmall deletions insertions polymorphisms and mutations[27]

16 Sequencing by Ligation The first high throughputsequencing by ligation was probably realized by Lynx Ther-apeutics Inc in their early technology of Massively ParallelSignature Sequencing [28] Churchrsquos laboratory at HarvardMedical School advanced the sequencing by ligation methodfor ultra fast and high throughput sequencing [13 14] Theyconverted an epifluorescence for rapid DNA sequencingDNA molecules were amplified in parallel onto micro beadsby emulsion polymerase chain reaction (Figure 1) Millionsof beads were immobilized in a polyacrylamide gel andsequenced using sequencing by ligation method AppliedBiosystem Inc acquired Agencourt Personal Genomics whodeveloped the Supported Oligo Ligation Detection system(SOLiD) for high throughput DNA sequencing

17 Bioinformatics Tools for Analyzing NGS Data Sequencereads generated from NGS technologies are shorter thantraditional Sanger sequences which makes assembly andanalysis of NGS data challenging Some important bioinfor-matics tools are available which is helpful in analyzing NGSdata (Table 1)

18 Applications of NGS Techniques NGS technologies havealready been used for various applications ranging from


TGG

TGG




Beforeamplification

SingleDNAmolecule

Polony PCRcolony

Afteramplification

Images of polonies

FISSEQ



Continue cycles

GCC

GCC

GCC

A

TGG

AC

C

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

(a)

Probes



anchor

Each time the


nucleotide

G

A

T

N N N NN N N N

N N N NN N N N

N N N NN N N N

N N N NN N N N

C

(b)

Extendedbases


New base being

incorporated

Cleavable cap

ChIP

Extend

Cleave


(c)





Gene to target

Target to SoC

SoC to candidate

FTIH to POC

POC to phase III

Phase III

Phase IV


Drug resistance

DiagnosticsPatient

stratification

Drug rescueRelease

Tran

scrip

tom

ics Gene

variation

Dise

ase

unde

rsta

ndin

g

Epigenetics

Protein interaction

Ab development

Mouse mutants

Encoded library

technology















2 Conclusion


Abbreviations






Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TGG

TGG




Beforeamplification

SingleDNAmolecule

Polony PCRcolony

Afteramplification

Images of polonies

FISSEQ



Continue cycles

GCC

GCC

GCC

A

TGG

AC

C

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

39984005998400

(a)

Probes



anchor

Each time the


nucleotide

G

A

T

N N N NN N N N

N N N NN N N N

N N N NN N N N

N N N NN N N N

C

(b)

Extendedbases


New base being

incorporated

Cleavable cap

ChIP

Extend

Cleave


(c)





Gene to target

Target to SoC

SoC to candidate

FTIH to POC

POC to phase III

Phase III

Phase IV


Drug resistance

DiagnosticsPatient

stratification

Drug rescueRelease

Tran

scrip

tom

ics Gene

variation

Dise

ase

unde

rsta

ndin

g

Epigenetics

Protein interaction

Ab development

Mouse mutants

Encoded library

technology















2 Conclusion


Abbreviations






Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Gene to target

Target to SoC

SoC to candidate

FTIH to POC

POC to phase III

Phase III

Phase IV


Drug resistance

DiagnosticsPatient

stratification

Drug rescueRelease

Tran

scrip

tom

ics Gene

variation

Dise

ase

unde

rsta

ndin

g

Epigenetics

Protein interaction

Ab development

Mouse mutants

Encoded library

technology















2 Conclusion


Abbreviations






Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










2 Conclusion


Abbreviations






Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article next generation sequencing: potential and...

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