宏基因组靶向测序分析皮肤表面微生物群落方法优化

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.016

生物技术通报 ›› 2016, Vol. 32 ›› Issue (11): 137-143.doi: 10.13560/j.cnki.biotech.bull.1985.2016.11.016

宏基因组靶向测序分析皮肤表面微生物群落方法优化

姚雪¹, 刘文丽², 裴广倩², 童贻刚², 罗亚平¹

1. 中国人民公安大学刑事科学技术学院,北京 100038;
2. 军事医学科学院微生物流行病研究所,北京 100071

收稿日期:2016-03-28 出版日期:2016-11-25 发布日期:2016-11-11
作者简介:姚雪,女,博士研究生,研究方向：刑事科学技术;E-mail：yaoxue1986@163.com
基金资助:
北京市科技新星计划交叉学科合作课题（xxhz201510）,上海市刑事科学技术研究院现场物证重点实验室开放课题（2014XCWZK14）

Optimization of Method for Analyzing Microbial Community on Human Skin by Targeted Sequencing of Metagenomics

YAO Xue¹, LIU Wen-li², PEI Guang-qian², TONG Yi-gang², LUO Ya-ping¹

1. School of Criminal Science and Technology,People's Public Security University of China, Beijing 100038;
2. Institute of Microbiology and Epidemiology,the Academy of Military Medical Sciences,Beijing 100071

Received:2016-03-28 Published:2016-11-25 Online:2016-11-11

摘要/Abstract

摘要： 通过实验设计和数据分析,筛选出尽可能真实反映含量较低的皮肤微生物群落的高通量测序分析方法。使用多因素多水平完全随机实验设计,分析DNA提取、PCR扩增、样品采集、测序过程中各种实验因素对微生物群落组成特征分析结果的影响。结果显示,在样品采集方面,筛选出了最佳的采集拭子与采集液体;在DNA提取方面,筛选出了最佳的前处理方法及试剂盒种类;在PCR反应方面,筛选出了最佳的模板浓度及退火温度;在测序方面,筛选出了合适的测序深度。通过一系列的实验条件筛选,确定了适合分析低含量细菌群落结构组成特征的方法,并成功地应用于人类手掌面皮肤。

关键词: 皮肤表面微生物群落, 宏基因组靶向测序, 16S rRNA基因

Abstract: Based on the experimental design and data analysis, we screened a high-throughput sequencing method which may measure the community of microorganisms in a low level on the human skin as real as possible. By completely random design of the experiments with multi-factor multi-level, we analyzed the effects of varied factors in the DNA extraction, PCR amplification, sample collection and sequencing on the composition characteristics of microbial community. The results showed that regarding the sample collection, the optimal collection swab and extraction liquid were screened out. In the aspect of DNA, the optimal pretreatment method and reagent box type were selected; on PCR reaction, the optimal template concentration and annealing temperature were determined; and on the sequencing aspect, the suitable sequencing depth was also chosen. In conclusion, through above series of experiments to screen the conditions, the appropriate method for analyzing the composition characteristics of community structure of bacteria in a low level was determined, and successfully applied to human palm skin.

Key words: microbial community on human skin, targeted sequencing of metagenomics, 16S rRNA gene

姚雪, 刘文丽, 裴广倩, 童贻刚, 罗亚平. 宏基因组靶向测序分析皮肤表面微生物群落方法优化[J]. 生物技术通报, 2016, 32(11): 137-143.

YAO Xue, LIU Wen-li, PEI Guang-qian, TONG Yi-gang, LUO Ya-ping. Optimization of Method for Analyzing Microbial Community on Human Skin by Targeted Sequencing of Metagenomics[J]. Biotechnology Bulletin, 2016, 32(11): 137-143.

参考文献

[1] Petrosino JF, Highlander S, Luna RA, et al. Metagenomic pyrosequencing and microbial identification[J]. Clinical Chemistry, 2009, 55（5）：856-866.
[2] Grice EA, Segre JA. The skin microbiome[J]. Nature Reviews Microbiology, 2011, 9（4）：244-253.
[3] Fierera N, Lauberb CL, Zhoub N, et al. Forensic identification using skin bacterial communities[J]. Proc Natl Acad Sci USA, 2010, 107（14）：6477-6481.
[4] Caporaso JG, Lauber CL, Costello EK, et al. Moving pictures of the human microbiome[J]. Genome Biology, 2011, 12：R50.
[5] Kueneman JG, Parfrey LW, Woodhams DC, et al. The amphibian skin-associated microbiome across species, space and life history stages[J]. Molecular Ecology, 2014, 23（6）：1238-1250.
[6] Tomic-Canic M, Perez-Perez GI, Blumenberg M. Cutaneous microbiome studies in the times of affordable sequencing[J]. Journal of Dermatological Science, 2014, 75（2）：82-87.
[7] Scharschmidt TC, Fischbach MA. What Lives on our skin：ecology, genomics and therapeutic opportunities of the skin microbiome[J]：Drug Discov Today Dis Mech, 2013, 10（3-4）. Pii：e83-e89.
[8] Blaser MJ, Dominguez-Bello MG, Contreras M, et al. Distinct cutaneous bacterial assemblages in a sampling of South American Amerindians and US residents[J]. International Society for Microbial Ecology, 2013, 7：85-95.
[9] Clemente JC, Pehrsson EC, et al. The microbiome of uncontacted Amerindians[J]. Sci Adv, 2015, 1（3）：pii：e1500183.
[10] Leung MHY, Wilkins DH and Lee P K. Insights into the pan-microbiome：skin microbial communities of Chinese individuals differ from other racial groups[J]. Sci Rep, 2015, 5：11845.
[11] Song SJ, Lauber C, Costello EK, et al. Cohabiting family members share microbiota with one another and with their dogs[J]. eLife. 2013, 2：e00458.
[12] Fierera N, Hamadyc M, et al. The influence of sex, handedness, and washing on the diversity of hand surface bacteria[J]. Proc Natl Acad Sci USA, 2008, 105（46）：17994-17999.
[13] Flores GE, Caporaso JG, Henley JB, et al. Temporal variability is a personalized feature of the human microbiome[J]. Genome Biology, 2014, 15：531.
[14] Meisel JS, Hannigan GD, Tyldsley AS, et al. Skin microbiome surveys are strongly influenced by experimental design[J]. J Invest Dermatol, 2016, 136（5）：2016. 01. 016.
[15] Huson DH, Richter DC, et al. Methods for comparative metageno-mics[J]. BMC Bioinformatics, 2009, 10（Suppl 1）：S12.
[16] 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, 75：7537-7541.
[17] Youssef N, Sheik CS, Krumholz LR, et al. Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys[J]. Applied and Environmental Microbiology, 2009, 75：5227-5236.
[18] Kennedy K, Hall MW, Lynch MDJ, et al. Evaluating bias of illumina-based bacterial 16S rRNA gene profiles[J]. Applied and Environmental Microbiology, 2014, 80（18）：5717-5722.
[19] Grice EA, Kong HH, Renaud G, et al. A diversity profile of the human skin microbiota[J]. Genome Res, 2008, 18：1043-1050.
[20] Ursell LK, Clemente JC, Rideout JR, et al. The interpersonal and intrapersonal diversity of human associated microbiota in key body sites[J]. J Allergy Clini Immunol, 2012, 129（5）：1204-1208.
[21] Liu Z, Lozupone C, Hamady M, et al. Short pyrosequencing reads suffice for accurate microbial community analysis[J]. Nucleic Acids Research, 2007, 35（18）：e120.
[22] Hamady M, Walker JJ, Harris JK, et al. Error-correcting barcoded primers allow hundreds of samples to be pyrosequenced in multiplex[J]. Nature Methods, 2008, 5（3）：235-237.

宏基因组靶向测序分析皮肤表面微生物群落方法优化

Optimization of Method for Analyzing Microbial Community on Human Skin by Targeted Sequencing of Metagenomics

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