基于三代测序技术的微生物组学研究进展
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摘要
微生物在人类生活中无处不在,过去人们对微生物的认识仅停留在单菌培养和定性研究上,而测序技术的发展极大地促进了微生物组学的研究。越来越多的证据表明:人体共生微生物、特别是肠道微生物与人类健康息息相关。二代测序技术凭借其高通量、高准确率和低成本的特点,成为微生物组学研究中的主流测序技术。但是随着研究的深入,二代测序技术的短读长(<450 bp)增加了后续数据分析和基因组拼接难度,也限制了该技术在未来研究中的应用。在此背景下,第三代测序技术应运而生。第三代测序技术又称单分子测序,能够直接对单个DNA分子进行实时测序,而不需要经过PCR扩增。第三代测序技术的平均读长在2–10 kb左右,最高可以达到2.2 Mb,实现了长序列的高通量测序。凭借其超长的测序读长、无GC偏好性等优势,三代测序技术为微生物基因组全长测序,组装完整可靠的基因组提供了新的方法。本文在描述三代测序的技术特点和原理的基础上,重点介绍了三代测序技术在微生物16S/18S rRNA基因测序、单菌的基因组组装以及宏基因组中的研究应用和进展。
Microbes are ubiquitous in human life.In years past,the study of microbes has only focused on single-bacteria cultures and qualitative analyses.The development of sequencing technology has greatly enhanced progress in microbiology research and more and more evidence shows that human symbiotic microbes,especially intestinal microbes,are closely related to human health.Second-generation sequencing technology is currently mainstream in microbiology research because of its high throughput,high accuracy and low cost.However,with the deepening complexity of research,the disadvantages of second-generation technology,i.e.short read length(< 450 bp),lead to subsequent challenges in data analysis and genome assembly,and limit the applicability to future research.In this context,the third-generation sequencing technology comes into being.The third-generation of sequencing(TGS) technology is also called single molecule sequencing.It directly carries out real-time sequencing of single DNA molecules without PCR amplifications.TGS technology significantly increases read length up to 2–10 kb or even 2.2 Mb.Because of its advantages of long read and no preference for GC,TGS provides a new method for full-length gene sequencing that facilitates the assembly of complete and reliable genome maps in microbes and that further reveals the diversity of microbial structures and functions.This review summarizes the technical characteristics and principles of TGS,and then mainly analyzes its applications and progress in 16 S/18 S rRNA gene sequencing,complete bacterial genome mapping and metagenomics research.
Microbes are ubiquitous in human life.In years past,the study of microbes has only focused on single-bacteria cultures and qualitative analyses.The development of sequencing technology has greatly enhanced progress in microbiology research and more and more evidence shows that human symbiotic microbes,especially intestinal microbes,are closely related to human health.Second-generation sequencing technology is currently mainstream in microbiology research because of its high throughput,high accuracy and low cost.However,with the deepening complexity of research,the disadvantages of second-generation technology,i.e.short read length(< 450 bp),lead to subsequent challenges in data analysis and genome assembly,and limit the applicability to future research.In this context,the third-generation sequencing technology comes into being.The third-generation of sequencing(TGS) technology is also called single molecule sequencing.It directly carries out real-time sequencing of single DNA molecules without PCR amplifications.TGS technology significantly increases read length up to 2–10 kb or even 2.2 Mb.Because of its advantages of long read and no preference for GC,TGS provides a new method for full-length gene sequencing that facilitates the assembly of complete and reliable genome maps in microbes and that further reveals the diversity of microbial structures and functions.This review summarizes the technical characteristics and principles of TGS,and then mainly analyzes its applications and progress in 16 S/18 S rRNA gene sequencing,complete bacterial genome mapping and metagenomics research.
引文
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Gloor GB,Hummelen R,Macklaim JM,Dickson RJ,Fernandes AD,MacPhee R,Reid G(2010)Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products.PLoS ONE,5,e15406.
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Huang ZR,Hong JL,Xu JX,Li L,Guo WL,Pan YY,Chen SJ,Bai WD,Rao PF,Ni L,Zhao LN,Liu B,Lv XC(2018)Exploring core functional microbiota responsible for the production of volatile flavour during the traditional brewing of Wuyi Hong Qu glutinous rice wine.Food Microbiology,76,487-496.
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Hugenholtz P,Pitulle C,Hershberger KL,Pace NR(1998)Novel division level bacterial diversity in a Yellowstone hot spring.Journal of Bacteriology,180,366-376.
Jain M,Koren S,Miga KH(2018)Nanopore sequencing and assembly of a human genome with ultra-long reads.Nature Biotechnology,36,338-345.
Jonasson J,Monstein HJ(2002)Classification,identification and subtyping of bacteria based on pyrosequencing and signature matching of 16s rDNA fragments.Apmis,110,263-272.
Liem M,Jansen HJ,Dirks RP,Henkel CV,van Heusden GPH,Lemmers R,Omer T,Shao S,Punt PJ,Spaink HP(2017)De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing.F1000Research,6,618.
Liu H,Hu Z,Zhang Y,Zhang J,Xie H,Liang S(2018)Microbial nitrogen removal of ammonia wastewater in poly(butylenes succinate)-based constructed wetland:Effect of dissolved oxygen.Applied Microbiology and Biotechnology,102,9389-9398.
Ludden C,Reuter S,Judge K,Gouliouris T,Blane B,Coll F,Naydenova P,Hunt M,Tracey A,Hopkins KL,Brown NM,Woodford N,Parkhill J,Peacock SJ(2017)Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing.Microbial Genomics,3,1-12.
Magi A,Semeraro R,Mingrino A,Giusti B,D’Aurizio R(2017)Nanopore sequencing data analysis:State of the art,applications and challenges.Briefings in Bioinformatics,19,1256-1272.
Mardis ER(2008)Next-generation DNA sequencing methods.Annual Review of Genomics and Human Genetics,9,387-402.
Metzker ML(2010)Sequencing technologies-The next generation.Nature Reviews Genetics,11,31-46.
Niedringhaus TP,Milanova D,Kerby MB,Snyder MP,Barron AE(2011)Landscape of next-generation squencing technologies.Analytical Chemistry,83,4327-4341.
Payne A,Holmes N,Rakyan V,Loose M(2018)Whale watching with BulkVis:A graphical viewer for Oxford Nanopore bulk fast5 files.bioRxiv,doi:https://doi.org/10.1101/312256.
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Schmid M,Frei D,Patrignani A,Schlapbach R,Frey JE,Remus-Emsermann MNP,Ahrens CH(2018)Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long,near identical repeats.Nucleic Acids Research,46,8953-8965.
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Theuns S,Vanmechelen B,Bernaert Q,Deboutte W,Vandenhole M,Beller L,Matthijnssens J,Maes P,Nauwynck HJ(2018)Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus.Scientific Reports,8,9830.
Tsai YC,Conlan S,Deming C,Segre JA,Kong HH,Korlach J,Oh J,Progra NCS(2016)Resolving the complexity of human skin metagenomes using single-molecule sequencing.mBio,7,e01748-15.
Wick RR,Judd LM,Gorrie CL,Holt KE(2017)Completing bacterial genome assemblies with multiplex MinION sequencing.Microbial Genomics,3,e000132.
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Benitez-Paez A,Portune KJ,Sanz Y(2016)Species-level resolution of 16S rRNA gene amplicons sequenced through the MinIONTM portable nanopore sequencer.GigaScience,5,4.
Brown SD,Nagaraju S,Utturkar S,De Tissera S,Segovia S,Mitchell W,Land ML,Dassanayake A,Kopke M(2014)Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia.Biotechnology for Biofuels,7,40.
Callaway E(2018)Flu virus finally sequenced in its native form.Nature,556,420.
Chin CS,Alexander DH,Marks P,Klammer AA,Drake J,Heiner C,Clum A,Copeland A,Huddleston J,Eichler EE,Turner SW,Korlach J(2013)Nonhybrid,finished microbial genome assemblies from long-read SMRT sequencing data.Nature Methods,10,563-569.
Clarke J,Wu HC,Jayasinghe L,Patel A,Reid S,Bayley H(2009)Continuous base identification for single-molecule nanopore DNA sequencing.Nature Nanotechnology,4,265-270.
Cusco A,Vines J,D’Andreano S,Riva F,Casellas J,Sánchez A,Francino O(2018)Using Min ION to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach.bioRxiv,doi:https://doi.org/10.1101/167015.
Eid 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 XX,Kuse R,Lacroix Y,Lin S,Lundquist P,Ma CC,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(2009)Real-time DNA sequencing from single polymerase molecules.Science,323,133-138.
Faino L,Seidl MF,Datema E,van den Berg GC,Janssen A,Wittenberg AH,Thomma BP(2015)Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome.mBio,6,e00936-15.
Fierer N,Breitbart M,Nulton J,Salamon P,Lozupone C,Jones R,Robeson M,Edwards RA,Felts B,Rayhawk S,Knight R,Rohwer F,Jackson RB(2007)Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria,archaea,fungi,and viruses in soil.Applied and Environmental Microbiology,73,7059-7066.
Frank JA,Pan Y,Tooming-Klunderud A,Eijsink VGH,McHardy AC,Nederbragt AJ,Pope PB(2016)Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.Scientific Reports,6,25373
Franzen O,Hu J,Bao X,Itzkowitz SH,Peter I,Bashir A(2015)Improved OTU-picking using long-read 16S r RNA gene amplicon sequencing and generic hierarchical clustering.Microbiome,3,43.
Gloor GB,Hummelen R,Macklaim JM,Dickson RJ,Fernandes AD,MacPhee R,Reid G(2010)Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products.PLoS ONE,5,e15406.
Greninger AL,Naccache SN,Federman S,Yu GX,Mbala P,Bres V,Stryke D,Bouquet J,Somasekar S,Linnen JM,Dodd R,Mulembakani P,Schneider BS,Muyembe-Tamfum JJ,Stramer SL,Chiu CY(2015)Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis.Genome Medicine,7,99.
Huang ZR,Hong JL,Xu JX,Li L,Guo WL,Pan YY,Chen SJ,Bai WD,Rao PF,Ni L,Zhao LN,Liu B,Lv XC(2018)Exploring core functional microbiota responsible for the production of volatile flavour during the traditional brewing of Wuyi Hong Qu glutinous rice wine.Food Microbiology,76,487-496.
Hug LA,Baker BJ,Anantharaman K,Brown CT,Probst AJ,Castelle CJ,Butterfield CN,Hernsdorf AW,Amano Y,Ise K,Suzuki Y,Dudek N,Relman DA,Finstad KM,Amundson R,Thomas BC,Banfield JF(2016)A new view of the tree of life.Nature Microbiology,1,16048.
Hugenholtz P,Pitulle C,Hershberger KL,Pace NR(1998)Novel division level bacterial diversity in a Yellowstone hot spring.Journal of Bacteriology,180,366-376.
Jain M,Koren S,Miga KH(2018)Nanopore sequencing and assembly of a human genome with ultra-long reads.Nature Biotechnology,36,338-345.
Jonasson J,Monstein HJ(2002)Classification,identification and subtyping of bacteria based on pyrosequencing and signature matching of 16s rDNA fragments.Apmis,110,263-272.
Liem M,Jansen HJ,Dirks RP,Henkel CV,van Heusden GPH,Lemmers R,Omer T,Shao S,Punt PJ,Spaink HP(2017)De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing.F1000Research,6,618.
Liu H,Hu Z,Zhang Y,Zhang J,Xie H,Liang S(2018)Microbial nitrogen removal of ammonia wastewater in poly(butylenes succinate)-based constructed wetland:Effect of dissolved oxygen.Applied Microbiology and Biotechnology,102,9389-9398.
Ludden C,Reuter S,Judge K,Gouliouris T,Blane B,Coll F,Naydenova P,Hunt M,Tracey A,Hopkins KL,Brown NM,Woodford N,Parkhill J,Peacock SJ(2017)Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing.Microbial Genomics,3,1-12.
Magi A,Semeraro R,Mingrino A,Giusti B,D’Aurizio R(2017)Nanopore sequencing data analysis:State of the art,applications and challenges.Briefings in Bioinformatics,19,1256-1272.
Mardis ER(2008)Next-generation DNA sequencing methods.Annual Review of Genomics and Human Genetics,9,387-402.
Metzker ML(2010)Sequencing technologies-The next generation.Nature Reviews Genetics,11,31-46.
Niedringhaus TP,Milanova D,Kerby MB,Snyder MP,Barron AE(2011)Landscape of next-generation squencing technologies.Analytical Chemistry,83,4327-4341.
Payne A,Holmes N,Rakyan V,Loose M(2018)Whale watching with BulkVis:A graphical viewer for Oxford Nanopore bulk fast5 files.bioRxiv,doi:https://doi.org/10.1101/312256.
Quick J,Loman NJ,Duraffour S,Simpson JT,Ettore S,Cowley L,Bore JA,Koundouno R,Dudas G,Mikhail A,Ouedraogo N,Afrough B,Bah A,Baum JHJ,Becker-Ziaja B,Boettcher JP,Cabeza-Cabrerizo M,Camino-Sanchez A,Carter LL,Doerrbecker J,Enkirch T,Garcia-Dorival I,Hetzelt N,Hinzmann J,Holm T,Kafetzopoulou LE,Koropogui M,Kosgey A,Kuisma E,Logue CH,Mazzarelli A,Meisel S,Mertens M,Michel J,Ngabo D,Nitzsche K,Pallasch E,Patrono LV,Portmann J,Repits JG,Rickett NY,Sachse A,Singethan K,Vitoriano I,Emanaberhan RLY,Zekeng EG,Racine T,Bello A,Sall AA,Faye O,Faye O,Magassouba N,Williams CV,Amburgey V,Winona L,Davis E,Gerlach J,Washington F,Monteil V,Jourdain M,Bererd M,Camara A,Somlare H,Camara A,Gerard M,Bado G,Baillet B,Delaune D,Nebie KY,Diarra A,Savane Y,Pallawo RB,Gutierrez GJ,Milhano N,Roger I,Williams CJ,Yattara F,Lewandowski K,Taylor J,Rachwal P,Turner DJ,Pollakis G,Hiscox JA,Matthews DA,O’Shea MK,Johnston AM,Wilson D,Hutley E,Smit E,Di Caro A,Wolfel R,Stoecker K,Fleischmann E,Gabriel M,Weller SA,Koivogui L,Diallo B,Keita S,Rambaut A,Formenty P,Gunther S,Carroll MW(2016)Real-time,portable genome sequencing for Ebola surveillance.Nature,530,228-232.
Sanger F,Nicklen S,Coulson AR(1977)DNA sequencing with chain-terminating inhibitors.Proceedings of the National Academy of Sciences,USA,74,5463-5467.
Schloss PD,Handelsman J(2005)Metagenomics for studying unculturable microorganisms:Cutting the Gordian knot.Genome Biology,6,229.
Schmid M,Frei D,Patrignani A,Schlapbach R,Frey JE,Remus-Emsermann MNP,Ahrens CH(2018)Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long,near identical repeats.Nucleic Acids Research,46,8953-8965.
Singer E,Bushnell B,Coleman-Derr D,Bowman B,Bowers RM,Levy A,Gies EA,Cheng JF,Copeland A,Klenk HP,Hallam SJ,Hugenholtz P,Tringe SG,Woyke T(2016)High-resolution phylogenetic microbial community profiling.ISME Journal,10,2020-2032.
Smith AM,Jain M,Mulroney L,Garalde DR,Akeson M(2017)Reading canonical and modified nucleotides in 16Sribosomal RNA using nanopore direct RNA sequencing.bioRxiv,doi:https://doi.org/10.1101/132274.
Sogin ML,Morrison HG,Huber JA,Mark Welch D,Huse SM,Neal PR,Arrieta JM,Herndl GJ(2006)Microbial diversity in the deep sea and the underexplored“rare biosphere”.Proceedings of the National Academy of Sciences,USA,103,12115-12120.
Sunagawa S,Coelho LP,Chaffron S,Kultima JR,Labadie K,Salazar G,Djahanschiri B,Zeller G,Mende DR,Alberti A,Cornejo-Castillo FM,Costea PI,Cruaud C,d’Ovidio F,Engelen S,Ferrera I,Gasol JM,Guidi L,Hildebrand F,Kokoszka F,Lepoivre C,Lima-Mendez G,Poulain J,Poulos BT,Royo-Llonch M,Sarmento H,Vieira-Silva S,Dimier C,Picheral M,Searson S,Kandels-Lewis S,Tara Oceans C,Bowler C,de Vargas C,Gorsky G,Grimsley N,Hingamp P,Iudicone D,Jaillon O,Not F,Ogata H,Pesant S,Speich S,Stemmann L,Sullivan MB,Weissenbach J,Wincker P,Karsenti E,Raes J,Acinas SG,Bork P(2015)Structure and function of the global ocean microbiome.Science,348,1261359.
Theuns S,Vanmechelen B,Bernaert Q,Deboutte W,Vandenhole M,Beller L,Matthijnssens J,Maes P,Nauwynck HJ(2018)Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus.Scientific Reports,8,9830.
Tsai YC,Conlan S,Deming C,Segre JA,Kong HH,Korlach J,Oh J,Progra NCS(2016)Resolving the complexity of human skin metagenomes using single-molecule sequencing.mBio,7,e01748-15.
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