基于SMRT测序技术的16S rRNA基因全长测序及其分析方法

doi:10.13560/j.cnki.biotech.bull.1985.2017-0036

摘要/Abstract

摘要： 被称为第三代测序技术的单分子测序是最近几年发展起来的高通量测序技术。其中，由Pacbio BioSciences公司开发的单分子实时测序技术（SMRT）是最先商用的技术。SMRT测序技术通过对模板序列循环测序产生环形一致序列（CCS），成功克服第三代测序技术准确率低的弊病。通过SMRT测序技术，科学家可以更深入准确地探究复杂环境微生物的结构和功能。介绍SMRT测序技术在微生物16S rRNA基因测序中的优势和劣势，并就基于SMRT测序技术所得的全长16S rRNA基因序列的质量控制、错误序列排除、聚类和注释分析等重要分析环节进行概述，同时，提出利用SMRT测序技术研究复杂环境微生物可能存在的问题及其解决方法，期望能为研究人员提供参考。

关键词: 单分子实时测序技术, PacBio RS Ⅱ, 第三代测序技术, 环形一致序列

Abstract: Single-molecule sequencing，called as third-generation sequencing technology，is a high-throughput technique developed in last few years. Of them，single-molecule real-time（SMRT）sequencing technology，developed by Pacific BioSciences（PacBio），is the first commercial technology. SMRT sequencing technology could successfully overcome the disadvantage of low accuracy in the third-generation sequencing technology，by generating circular consensus sequence（CCS）through cycle sequencing the template sequence. Therefore，SMRT sequencing technology will allow scientists to profoundly and accurately study the structures and functions of microbial communities in complex environment. Here，we introduced the advantages and disadvantages of SMRT sequencing technology in 16S rRNA gene sequence of microorganism，and summarized the important steps，such as quality control，filtering of error tags，clustering analysis，annotation analysis，etc. of full-length 16S rRNA gene sequence acquired by SMRT sequencing technology. In addition，we pointed out the problems and the feasible solutions while applying SMRT sequencing technology in the study of microbial in complex environment，aiming at providing references for researchers in this field.

Key words: single-molecule real-time sequencing technology, PacBio RS II, third-generation sequencing technology, circular consensus sequence

唐勇, 刘旭. 基于SMRT测序技术的16S rRNA基因全长测序及其分析方法[J]. 生物技术通报, 2017, 33(8): 34-39.

TANG Yong , LIU Xu. Full-length Sequencing of 16S rRNA Gene and Its Analysis Based on the SMRT Sequencing Technology[J]. Biotechnology Bulletin, 2017, 33(8): 34-39.

参考文献

[1] Klappenbach JA, Saxman PR, Cole JR, et al. rrndb：the ribosomal RNA operon copy number database[J] . Nucleic Acids Research, 2001, 29（1）：181-184.
[2] Sogin ML, Morrison HG, Huber JA, et al. Microbial diversity in the deep sea and the underexplored “rare biosphere”[J] . Proceedings of the National Academy of Sciences, 2006, 103（32）：12115-12120.
[3] Roh SW, Abell GC, Kim K-H, et al. Comparing microarrays and next-generation sequencing technologies for microbial ecology research[J] . Trends in Biotechnology, 2010, 28（6）：291-299.
[4] Margulies M, Egholm M, Altman WE, et al. Genome sequencing in microfabricated high-density picolitre reactors[J] . Nature, 2005, 437（7057）：376-380.
[5] Bentley DR. Whole-genome re-sequencing[J] . Current Opinion in Genetics & Development, 2006, 16（6）：545-552.
[6] Clarke J, Wu H-C, Jayasinghe L, et al. Continuous base identification for single-molecule nanopore DNA sequencing[J] . Nature Nanotechnology, 2009, 4（4）：265-270.
[7] McCarthy A. Third generation DNA sequencing：pacific biosciences’ single molecule real time technology[J] . Chemistry & Biology, 2010, 17（7）：675-676.
[8] Mikheyev AS, Tin MM. A first look at the Oxford Nanopore MinION sequencer[J] . Molecular Ecology Resources, 2014, 14（6）：1097-1102.
[9] Chakravorty S, Helb D, Burday M, et al. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria[J] . Journal of Microbiological Methods, 2007, 69（2）：330-339.
[10] Kim M, Morrison M, Yu Z. Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes[J] . J Microbiol Methods, 2011, 84（1）：81-87.
[11] Rhoads A, Au KF. PacBio sequencing and its applications[J] . Genomics, Proteomics & Bioinformatics, 2015, 13（5）：278-289.
[12] Roberts RJ, Carneiro MO, Schatz MC. The advantages of SMRT sequencing[J] . Genome Biology, 2013, 14（7）：405.
[13] Mosher JJ, Bowman B, Bernberg EL, et al. Improved performance of the PacBio SMRT technology for 16S rDNA sequencing[J] . Journal of Microbiological Methods, 2014, 104：59-60.
[14] Benitez-Paez A, Portune KJ, Sanz Y. Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION^TM portable nanopore sequencer[J] . Gigascience, 2016, 5（1）：1-9.
[15] Schloss PD, Jenior ML, Koumpouras CC, et al. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system[J] . PeerJ, 2016, 4：e1869.
[16] Lee CH, Bowman B, Hall R, et al. Developments in PacBio? metagenome sequencing：Shotgun whole genomes and full-length 16S[C] . International Plant and Animal Genome Conference Asia, 2014.
[17] Laver T, Harrison J, O’Neill P, et al. Assessing the performance of the Oxford Nanopore Technologies MinION[J] . Biomolecular Detection and Quantification, 2015, 3：1-8.
[18] Koren S, Schatz MC, Walenz BP, et al. Hybrid error correction and de novo assembly of single-molecule sequencing reads[J] . Nature Biotechnology, 2012, 30（7）：693-700.
[19] Ross MG, Russ C, Costello M, et al. Characterizing and measuring bias in sequence data[J] . Genome Biology, 2013, 14（5）：1.
[20] Eid J, Fehr A, Gray J, et al. Real-time DNA sequencing from single polymerase molecules[J] . Science, 2009, 323（5910）：133-138.
[21] Quail MA, Smith M, Coupland P, et al. A tale of three next generation sequencing platforms：comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers[J] . BMC Genomics, 2012, 13（1）：341.
[22] Patel RK, Jain M. NGS QC Toolkit：a toolkit for quality control of next generation sequencing data[J] . PLoS One, 2012, 7（2）：e30619.
[23] Andrews, S. FastQC：a quality control tool for high throughput sequence data[EB] . http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
[24] Bowman B, Shin MY, Lee JE, et al. Analysis of full-length metagenomic 16S genes by SMRT^? sequencing[J] . Chemistry, 2013, 4：C2.
[25] Edgar RC, Haas BJ, Clemente JC, et al. UCHIME improves sensitivity and speed of chimera detection[J] . Bioinformatics, 2011, 27（16）：2194-2200.
[26] Haas BJ, Gevers D, Earl AM, et al. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons[J] . Genome Research, 2011, 21（3）：494-504.
[27] Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project：improved data processing and web-based tools[J] . Nucleic Acids Research, 2013, 41（D1）：D590-D596.
[28] Maidak BL, Cole JR, Lilburn TG, et al. The RDP-II（ribosomal database project）[J] . Nucleic Acids Research, 2001, 29（1）：173-174.
[29] DeSantis TZ, Hugenholtz P, Larsen N, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB[J] . Applied and Environmental Microbiology, 2006, 72（7）：5069-5072.
[30] Bowman JS, Rasmussen S, Blom N, et al. Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene[J] . The ISME Journal, 2012, 6（1）：11-20.
[31] Tedersoo L, Nilsson RH, Abarenkov K, et al. 454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases[J] . New Phytologist, 2010, 188（1）：291-301.
[32] Lücking R, Lawrey JD, Gillevet PM, et al. Multiple ITS haplotypes in the genome of the lichenized basidiomycete Cora inversa（Hygrophoraceae）：fact or artifact？[J] . Journal of Molecular Evolution, 2014, 78（2）：148-162.
[33] Unterseher M, Jumpponen A, ?pik M, et al. Species abundance distributions and richness estimations in fungal metagenomics-lessons learned from community ecology[J] . Molecular Ecology, 2011, 20（2）：275-285.
[34] Kunin V, Engelbrektson A, Ochman H, et al. Wrinkles in the rare biosphere：pyrosequencing errors can lead to artificial inflation of diversity estimates[J] . Environmental Microbiology, 2010, 12（1）：118-123.
[35] Edgar RC. UPARSE：highly accurate OTU sequences from microbial amplicon reads[J] . Nature Methods, 2013, 10（10）：996-998.
[36] Schloss PD, Westcott SL, Ryabin T, et al. Introducing mothur：open-source, platform-independent, community-supported software for describing and comparing microbial communities[J] . Applied and Environmental Microbiology, 2009, 75（23）：7537-7541.
[37] Wang Q, Garrity GM, Tiedje JM, et al. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J] . Applied and Environmental Microbiology, 2007, 73（16）：5261-5267.
[38] Liu Z, DeSantis TZ, Andersen GL, et al. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers[J] . Nucleic Acids Research, 2008, 36（18）：e120.
[39] Burke CM, Darling AE. A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq[J] . Peer J, 2016, 4：e2492.
[40] Langille MG, Zaneveld J, Caporaso JG, et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences[J] . Nature Biotechnology, 2013, 31（9）：814-821.