三代测序技术及其应用研究进展

doi:10.16431/j.cnki.1671-7236.2019.08.007

摘要/Abstract

摘要：

通过DNA测序不仅可以更好地认识生命的本质，了解生物的差异性、进化及发展史，而且为重组DNA的研究提供了方向，在疾病诊断及基因分型方面具有非常重要的实用价值。首先从发现DNA是遗传物质开始，简要回顾了第一、二和三代测序技术的整个发展历程，以及各自的优缺点和主要测序技术或平台；其次，重点介绍了由PacBio公司开发的第三代测序典型技术——单分子实时测序技术（SMRT）和ONT纳米孔测序技术的原理、方法和技术流程，并总结了第三代测序技术的应用领域，包括基因组重测序、do novo测序、转录组研究、甲基化检测、与疾病相关的结构变异检测，以及流行病学中病毒准种分析等应用；再次，比较了三代测序技术在转录组测序和表观遗传学研究中的技术优势，第三代测序技术在基因组重复区域或结构变异等研究领域具有非常明显的优势，对多种疾病的研究意义重大，已成为未来重要的精准诊断工具，此外，凭借对重要经济物种遗传信息的解析，育种工作者通过建立基因与性状的关联，对持有优良性状基因的个体进行人工选育，大大缩短了育种年限；最后，对第三代测序技术在医疗、农业、环境等领域的应用前景进行了展望。

关键词: 第三代测序; 单分子测序; 长读长; 结构变异

Abstract:

DNA sequencing not only helps us better to understand the nature of life, biological differences, different biological evolutions and developmental history of organisms, but also provides direction for recombinant DNA research, and has very important practical value in disease diagnosis and genotyping.Firstly, from the discovery that DNA is a genetic material, we briefly review the entire development process of the first, second and third generation sequencing technologies, their respective advantages and disadvantages, and the main sequencing technologies or platforms in each.Secondly, we focus on the third generation sequencing typical technology developed by PacBio, including single molecule Real time (SMRT), Oxford Nanopore Technologies (ONT), introduce the principles, methods and technical processes, and summarize the application areas of the third generation sequencing technology, including the genomic resequencing, do novo sequencing, transcriptome studies, methylation testing, disease-related structural variation testing and the application of viral quasispecies analysis in epidemiology.Thirdly, the advantages of three generations of sequencing techniques in transcriptome sequencing and epigenetics are compared, the third generation sequencing technology has obvious advantages in research fields, such as genomic repeat regions or structural variations, and very meaningful for the study of various diseases, it is considered to be an important and accurate diagnostic tool in the future.In addition, breeders can artificially select individuals by establishing associations between genes and traits by analyzing the genetic information of important economic species, which greatly shortens the breeding period.Finally, we prospect the future application of the third generation sequencing technology in medicine, agriculture and environment.

Key words: the third generation sequencing; single molecule sequencing; long reads; structural variations

中图分类号:

马丽娜, 杨进波, 丁逸菲, 李颖康. 三代测序技术及其应用研究进展[J]. 《中国畜牧兽医》, 2019, 46(8): 2246-2256.

MA Lina, YANG Jinbo, DING Yifei, LI Yingkang. Research Progress on Three Generations Sequencing Technology and Its Application[J]. , 2019, 46(8): 2246-2256.

参考文献 52

[1]	SANGER F,COULSON A R.A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase[J]. Journal of Molecular Biology,1975,94(3):441-448.
[2]	SANGER F,NICKLEN S,COULSON A R.DNA sequencing with chain-terminating inhibitors[J]. Proceedings of the National Academy of Sciences,1977,74(12):5463-5467.
[3]	SANGER F,AIR G M,BARRELL B G,et al.Nucleotide sequence of bacteriophage phi X174 DNA[J]. Nature,1977,265(5596):687-695.
[4]	MAXAM A M,GILBERT W.A new method for sequencing DNA[J]. Proceedings of the National Academy of Sciences,1977,74(2):560-564.
[5]	SCHUSTER S C.Next-generation sequencing transforms today's biology[J]. Nature Methods,2008,5(1):16-18.
[6]	马文丽.基因测序实验技术[M].北京:化学工业出版社,2012. MA W L.The Experimental Technology of Gene Sequencing[M].Beijing:Chemical Industry Press,2012.(in Chinese)
[7]	ANSORGE W J.Next-generation DNA sequencing techniques[J]. New Biotechnology,2009,25(4):195-203.
[8]	LANDER E S,LINTON L M,BIRRRN B,et al.Initial sequencing and analysis of the human genome[J]. Nature,2001,409(6822):860-921.
[9]	VENTER J C,ADAMS M D,MYERS E W,et al.The sequence of the human genome[J]. Science,2001,291(5507):1304-1351.
[10]	SHENDURE J,JI H.Next-generation DNA sequencing[J]. Nature Biotechnology,2008,26(10):1135-1145.
[11]	MARDIS E R.Next-generation DNA sequencing methods[J]. Annual Review of Genomics and Human Genetics,2008,9:387-402.
[12]	METZKER M L.Sequencing technologies the next generation[J]. Nature Reviews Genetics,2010,11(1):31-46.
[13]	ROTHBERG J M,HINZ W,REARICK T M,et al.An integrated semiconductor device enabling nonoptical genome sequencing[J]. Nature,2011,475(7356):348-352.
[14]	BUERMANS H P,DEN DUNEN J T.Next generation sequencing technology:Advances and applications[J]. Biochimica et Biophysica Acta-Biomembranes,2014,1842(10):1932-1941.
[15]	RHOADS A,AU K F.PacBio sequencing and its applications[J]. Genomics Proteomics Bioinformatics,2015,13(5):278-289.
[16]	EID J,FEHR A,GRAY J,et al.Real-time DNA sequencing from single polymerase molecules[J]. Science,2009,323(5910):133-138.
[17]	FENG Y,ZHANG Y,YING C,et al.Nanopore-based fourth-generation DNA sequencing technology[J]. Genomics Proteomics Bioinformatics,2015,13(1):4-16.
[18]	LU H,GIORDANO F,NING Z.Oxford Nanopore MinION sequencing and genome assembly[J]. Genomics Proteomics Bioinformatics,2016,14(5):265-279.
[19]	乌日拉嘎,徐海燕,冯淑贞,等.测序技术的研究进展及三代测序的应用[J].中国乳品工业,2016,44(4):33-37. WURILAGA,XU H Y,FENG S Z,et al.Research progress of sequencing technologies and the application of third generation sequencing[J]. China Dairy Industry,2016,44(4):33-37.(in Chinese)
[20]	MCCARTHY A.Third generation DNA sequencing:Pacific biosciences' single molecule Real time technology[J]. Chemistry & Biology,2010,17(7):675-676.
[21]	ZHANG X,DAVENPORT K W,GU W,et al.Improving genome assemblies by sequencing PCR products with PacBio[J]. Biotechniques,2012,53(1):61-62.
[22]	FERRARINI M,MORETTO M,WARD J A,et al.An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome[J]. BMC Genomics,2013,14(1):670.
[23]	GORDON D,HUDDLESTON J,CHAISSPN M J,et al.Long-read sequence assembly of the gorilla genome[J]. Science,2016,352(6281):aae0344.
[24]	TYSON J R,ONEIL N J,JAIN M,et al.MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome[J]. Genome Research,2018,28:266-274.
[25]	PENG Z,HU Y,XⅡ J,et al.Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens[J]. BMC Genomics,2016,17(1):21.
[26]	SHI L,GUO Y,DONG C,et al.Long-read sequencing and de novo assembly of a Chinese genome[J]. Nature Communications,2016,7:12065.
[27]	CHAISSON M J,HUDDLESTON J,DENNIS M Y,et al.Resolving the complexity of the human genome using single-molecule sequencing[J]. Nature,2015,517(7536):608-611.
[28]	GUO L,WINZER T,YANG X,et al.The opium poppy genome and morphinan production[J]. Science,2018,362(6412):343-347.
[29]	CHIN C,PELUSO P,SEDLAZECK F J,et al.Phased diploid genome assembly with single molecule Real-time sequencing[J]. Nature Methods,2016,13:1050-1054.
[30]	MERKER J D,WENGER A M,SNEDDON T,et al.Long-read genome sequencing identifies causal structural variation in a Mendelian disease[J]. Genetics in Medicine,2018,20(1):159-163.
[31]	DOI K,MONJO T,HOANG P H,et al.Rapid detection of expanded short tandem repeats in personal genomics using hybrid sequencing[J]. Bioinformatics,2014,30(6):815-822.
[32]	ISHIURA H.Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy[J]. Nature Genetics,2018,50(4):581-590.
[33]	ZENG S,ZHANG M Y,WANG X J,et al.Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy[J]. Journal of Medical Genetics,2019,56(4):265-270.
[34]	LOOMIS E W,EID J S,PELUSO P,et al.Sequencing the unsequenceable:Expanded CGG-repeat alleles of the fragile X gene[J]. Genome Research,2013,23(1):121-128.
[35]	HARON D,TILGNER H,GRUBRRT F,et al.A single-molecule long-read survey of the human transcriptome[J]. Nature Biotechnology,2013,31(11):1009-1014.
[36]	WANG B,TSENG E,REGULSKI M,et al.Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing[J]. Nature Communications,2016,7:11708.
[37]	FLEMINH M B,PATTERSON E L,REEVES P A,et al.Exploring the fate of mRNA in aging seeds:Protection,destruction,or slow decay[J]. Journal of Experimental Botany,2018,69(18):4309-4321.
[38]	TILGNER H,GRUBERT F,SHARON D,et al.Defining a personal,allele-specific,and single-molecule long-read transcriptome[J]. Proceedings of the National Academy of Sciences,2014,111(27):9869-9874.
[39]	MURRAY I A,CLARK T A,MORGAN R D,et al.The methylomes of six bacteria[J]. Nucleic Acids Research,2012,40(22):11450-11462.
[40]	FLUSBERG B A,WEBSTER D R,LEE J H,et al.Direct detection of DNA methylation during single-molecule,Real-time sequencing[J]. Nature Methods,2010,7(6):461-465.
[41]	BECKMANN N D,KARRI S,FANG G,et al.Detecting epigenetic motifs in low coverage and metagenomics settings[J]. BMC Bioinformatics,2014,15(Suppl 9):S16.
[42]	SATOU K,SHIROMA A,TERUYA K,et al.Complete genome sequences of eight Helicobacter pylori strains with different virulence factor genotypes and methylation profiles,isolated from patients with diverse gastrointestinal diseases on Okinawa Island,Japan,determined using PacBio single-molecule Real-time technology[J]. Genome Announcement,2014,2(2):e00286-14.
[43]	WESCOE Z L,SCHREIBER J,AKESON M.Nanopores discriminate among five C5-cytosine variants in DNA[J]. Journal of the American Chemical Society,2014,136(47):16582-16587.
[44]	YAO B,CHENG Y,WANG Z,et al.N(6)-methyladenine DNA modification in the human genome[J]. Molecular Cell,2018,71(2):306-318.
[45]	TAKEDA H,UEDA Y,INUZUKA T,et al.Evolution of multi-drug resistant HCV clones from pre-existing resistant-associated variants during direct-acting antiviral therapy determined by third-generation sequencing[J]. Scientific Reports,2017,7:45605.
[46]	崔治中.病毒准种多样性及其在免疫选择压作用下的演变[J].生命科学,2013,25(9):843-852. CUI Z Z.Multiplicity of viral quasi species and its evolution under selective pressure of antibody immunity[J]. Chinese Bulletin of Life Sciences,2013,25(9):843-852.(in Chinese)
[47]	BASHIR A,KLAMMER A A,ROBINS W P,et al.A hybrid approach for the automated finishing of bacterial genomes[J]. Nature Biotechnology,2012,30(7):701-707.
[48]	JAIN M,KOREN S,MIGA K H,et al.Nanopore sequencing and assembly of a human genome with ultra-long reads[J]. Nature Biotechnology,2018,36(4):338-345.
[49]	KOREN S,PHILLIPPY A M.One chromosome,one contig:Complete microbial genomes from long-read sequencing and assembly[J]. Current Opinion in Microbiology,2015,23:110-120.
[50]	EISENSTEIN M.An ace in the hole for DNA sequencing[J]. Nature,2017,550(7675):285-288.
[51]	SANCHIS-JUAN A,STEPHENS J,FRENCH C E,et al.Complex structural variants resolved by short-read and long-read whole genome sequencing in mendelian disorders[J]. Genome Medicine,2018,10(1):95.
[52]	COUNCIL N R.Toward Precision Medicine:Building a Knowledge Network for Biomedical Research and a New Taxonomy of Disease[M].Washington D.C.:National Academies Press,2011.