The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies.
The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies.
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DOI：
10.1186/s12866-015-0351-6
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发表时间：
2015-03-21
期刊：
影响因子：
4.2
通讯作者：
Buck GA
中科院分区：
文献类型：
--
作者：
Brooks JP;Edwards DJ;Harwich MD Jr;Rivera MC;Fettweis JM;Serrano MG;Reris RA;Sheth NU;Huang B;Girerd P;Vaginal Microbiome Consortium;Strauss JF 3rd;Jefferson KK;Buck GA
表征微生物群落通过下一代测序是受到一些陷阱涉及样品处理。观察到的群落组成可能严重扭曲了微生物组中实际存在的细菌数量，阻碍了分析并威胁到宏基因组研究结论的有效性。我们介绍了一种实验协议，使用模拟社区来量化和表征样品处理管道中引入的偏差。我们使用了80个细菌模拟群落，由7种阴道相关菌株的规定比例的细胞组成，以评估样品处理管道中引入的偏差。通过混合规定数量的DNA和PCR产物，我们创建了另外两组80个模拟群落，以量化(1)DNA提取，(2)PCR扩增，(3)测序和分类分类对每个步骤的特定选择的偏差的相对贡献。我们根据观察到的比例开发了模型来预测环境样本的“真实”成分，并将其应用于四次访问期间来自单个受试者的一组临床阴道样本。我们观察到，使用不同的DNA提取试剂盒可以产生显着不同的结果，但无论选择哪种试剂盒，都会引入偏差。在一些样品中，我们观察到偏差的错误率超过85%，而大多数细菌的技术变异非常低，小于5%。DNA提取和PCR扩增对我们方案的影响远大于测序和分类。处理步骤以不同的方式影响不同的细菌，导致一个群落的观察比例放大和抑制。当预测模型应用于受试者的临床样本时，预测的微生物组谱比观察到的群落组成更能反映受试者在就诊时的生理和诊断。尽管测序技术取得了进一步的进步，但由于DNA提取和PCR扩增而导致的16S研究中的偏倚仍然需要引起关注。对模拟群落的分析可以帮助评估偏见，并促进对环境样本结果的解释。本文的在线版本（doi:10.1186/s12866-015-0351-6）包含补充材料，可供授权用户使用。
Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits. We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions. Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples. The online version of this article (doi:10.1186/s12866-015-0351-6) contains supplementary material, which is available to authorized users.
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影响因子：
5.7
作者：
Lagier JC;Million M;Hugon P;Armougom F;Raoult D
通讯作者：
Raoult D
影响因子：
46.9
作者：
Knight R;Jansson J;Field D;Fierer N;Desai N;Fuhrman JA;Hugenholtz P;van der Lelie D;Meyer F;Stevens R;Bailey MJ;Gordon JI;Kowalchuk GA;Gilbert JA
通讯作者：
Gilbert JA
影响因子：
4.4
作者：
Fettweis JM;Serrano MG;Sheth NU;Mayer CM;Glascock AL;Brooks JP;Jefferson KK;Vaginal Microbiome Consortium (additional members);Buck GA
通讯作者：
Buck GA
影响因子：
4.4
作者：
Lauber, Christian L.;Hamady, Micah;Fierer, Noah
通讯作者：
Fierer, Noah
影响因子：
4.4
作者：
Harwich MD Jr;Serrano MG;Fettweis JM;Alves JM;Reimers MA;Vaginal Microbiome Consortium (additional members);Buck GA;Jefferson KK
通讯作者：
Jefferson KK