新一代测序技术的发展及应用前景

生物技术通报BIOTECHNOLOGYBULLETIN201010杨晓玲施苏华唐恬(,510275):Sanger测序的提出给人类破译遗传密码提供了强有力的工具而近几年飞速发展起来的高通量测序技术无论在成本还是效率上,都对传统测序提出了巨大的挑战千元人类基因组计划的公布更是加快了测序研发的进程通过下一代测序技术,科学家不仅能够绘制出各个物种的基因组图谱,还能探讨不同条件下的基因表达差异以及不同物种之间或者物种之内的序列差异,进而为传统生物学以及医学研究开辟新的视角与此同时,随着各大测序平台的不断改进,普通实验室都有能力承担此类大型测序项目就目前正在发展的几种高通量测序技术进行了综合阐述,并比较了各种测序技术的优缺点,最后对其应用领域作了简单介绍:第二代测序第三代测序基因组转录组重测序ResearchProgressandApplicationofNextgenerationSequencingYangXiaolingShiSuhuaTangTian(SchoolofLifeSciences,SunYatsenUniversity,Guangzhou510275)Abstrac:tThedevelopmentofSangersequencinghasbroughtapowerfultoolforhumanbeingtodecipherthegeneticcode.However,thearrivalofhighthroughputsequencingoverthepastseveralyearshighlychallengesthetraditionalmethod,notonlyincostbutalsoinefficiency.Aracetowardthe$1000genomedramaticallyacceleratesthespeedoftechnologicalinnovation.Therefore,nextgenerationsequencingwillallowustodrawacompletegenomemap,comparegeneexpressionunderdifferentconditions,anddemonstratesequencevariationbetweenorwithinspecies,allofwhichwouldopenoutanewavenueforbiologicalormedicalresearch.Atthesametime,withthehighlydevelopedsequencingplatforms,nearlyeveryresearchteamwouldhavetheopportunitytocarryoutsuchresearch.Inthispaper,weprovideasnapshotofthesenewapproaches,discusstheadvantagesandlimitationsofthem,andalsoillustratetheirapplicationsinvariousfields.Keywords:SecondgenerationsequencingThirdgenerationsequencingGenomeTranscriptomeResequencing:20100412:(30730008,30970208,40976081),973!(2007CB815703),,,:,,,:;Emai:[e-mail]:,,Emai:[e-mail],,[1]1977SangerCoulson[1,2],2010,;,,,,,,1Sanger30DNA,,,,Sanger,,201010:1.1第二代测序∀∀∀高通量低成本齐头并进,,[3]:1.1.1Roche454Sanger,,,454[4],,DNA,20104,700454(),IlluminaSolexaDNA,Solexa[5]454,Solexa,,,Solexa2008,,400()1.1.22007ABIChurch[6]SOLiD[7],,,SNP,4,,SOLiD3.020G,35-50bp,Solexa,ABI,DanaherMotionPolonator[8]Church,,CompleteGenomicsDNA[9],,31.2第三代测序∀∀∀单分子长片段有望实现,PCRPCR,DNAHeliscope,,Helicos[10],,,PacificBiosciencesDNA,DNA,DNA[11,12],,2Kahvejian2008[13]:,?!,,,DNA,,,,,2.1DNA水平的应用2.1.177生物技术通报BiotechnologyBulletin201010,[14,15],,DNA,Rasmusse[16]4000DNA,Solexa,79%,,2.1.2,,,[17],Feuk[18],,Trick[19]Solexa,2-4SNP,;,DNA[20];,SNP2.1.3DNA,,,,,(MetaHIT)[21],,2.2RNA水平的应用2.2.1,,,,,,SNPCloonan[22]SOLiD,,;Tang[23]RNASeq,,75%,8%-19%2.2.2RNARNA,,,,,RNA,RNA,miRNA,miRNA,miRNA,miRNARNALu[24]4543miRNA,miRNA,!!;Morin[25]SolexaRNA,81RNA,23miRNA2.3表观基因组学应用2.3.1,DNA,DNA,-(ChIPseq)[26]Robertson[27]STAT1,,ChIPPCR,2.3.2DNADNA,,DNADNA,78201010:Taylor[28]454,;Barski[29]Solexa20,,3(1),,GAP,,DNA,454,,,[30]Solexa454,miRNASolexa(pairedendsequencing),,,ATSOLiD,SNP,,,,Goldberg[32]Sanger454,,,,,SNP[30]1[412,3033](bp)(%)(/MB)3730XL0.2M800-100099.992500DNA454400M400-5009940()SolexaGAII8-10G75992()Hi-Seq200G100SOLiDSOLiDTM320G25982()SOLiDTM4100G50Polonator4-5G13981()CompleteGenomics(DNA)Helicos35G3296.41PacificNanopore44.1测序费用直线下降,千元基因组计划指日可待2006,TheXPrizeFoundation1[30],1000,ABI3730,100,10,,79生物技术通报BiotechnologyBulletin2010101000100002030,10002009,CompleteGenomics,4400[34],4.2序列长度限制拼接,数据质量有待提高,,,Sanger,Sanger,,,,Sanger25bp,,750bp1000bp;454100bpGSFLX250bp,400bpDNA,4.3样品准备步骤繁琐,文库构建仍需简化,,,,,,,,,,RNA,,,,,RNAseq,[35],RNA,,,PCR,4.4海量数据提出挑战,信息平台尚未完善,,,,;,,,,,,,AMOScmp,90%[36],,,,,,NCBI(theSequenceReadArchive,SRA),[1]SangerF,AirGM,BarrellBG,eta.lNucleotidesequenceofbacteriophagephiX174DNA.Nature,1977,265(5596):687695.[2]SangerF,NicklenS,CoulsonAR.DNAsequencingwithchainterminatinginhibitors.ProcNalAcadSciUSA,1977,74(12):54635467.[3]AksyonovSA,BittnerM,BloomLB,eta.lMultiplexedDNAsequencingbysynthesis.AnnualReviewofBiochemistry,2006,348(1):12738.[4]RothbergJM,LeamonJH.Thedevelopmentandimpactof454sequencing.NatureBiotechnology,2008,26(10):111724.[5]TurcattiG,RomieuA,FedurcoM,TairiAP.Anewclassofcleavablefluorescentnucleotides:synthesisandoptimizationasreversibleterminatorsforDNAsequencingbysynthesis.NucleicAcidResearchJourna,l2008,36(4):e25.[6]ShendureJ,PorrecaGJ,ReppasNB,eta.lAccuratemultiplexpolonysequencingofanevolvedbacterialgenome.Science,2005,309(5741):172832.[7]McKernanKJ,PeckhamHE,CostaGL,eta.lSequenceandstructuralvariationinahumangenomeuncoveredbyshortread,massivelyparallelligationsequencingusingtwobaseencoding.GenomeResearch,2009,19(9):152741.[8]PolonatorFrequentlyAskedQuestions.:faqs.aspx[9]DrmanacR,SparksAB,CallowMJ,eta.lHumangenomesequencingusingunchainedbasereadsonselfassemblingDNAnanoarrays.Science,2010,327(5961):7881.[10]HarrisTD,BuzbyPR,BabcockH,eta.lSinglemoleculeDNAsequencingofaviralgenome.Science,2008,320(5872):1069.[11]EidJ,FehrA,GrayJ,eta.lRealtimeDNAsequencingfromsinglepolymerasemolecules.Science,2009,323(5910):1338.[12]RuskN.Cheapthirdgenerationsequencing.NatureMethods,2009,6(4):244.[13]KahvejianA,QuackenbushJ,ThompsonJF.Whatwouldyoudoifyoucouldsequenceeverything.NatureBiotechnology,2008,26(10):112533.[14]LiR,FanW,TianG,eta.lThesequenceanddenovoassemblyofthegiantpandagenome.Nature,2010,463(7279):3117.[15]HuangS,Li