Quantification of variation and the impact of biomass in targeted 16S rRNA gene sequencing studies.
Quantification of variation and the impact of biomass in targeted 16S rRNA gene sequencing studies.
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DOI：
10.1186/s40168-018-0543-z
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发表时间：
2018-09-10
期刊：
影响因子：
15.5
通讯作者：
Aldrovandi GM
中科院分区：
文献类型：
--
作者：
Bender JM;Li F;Adisetiyo H;Lee D;Zabih S;Hung L;Wilkinson TA;Pannaraj PS;She RC;Bard JD;Tobin NH;Aldrovandi GM
测序技术和生物信息学工具的最新进展使大规模的微生物组研究成为可能，这些研究正在迅速推进医学研究。然而，技术或分析的微小变化可能会显著改变结果，并导致相互矛盾的结果。量化靶向16 S rRNA基因测序研究中预期的技术与生物学变异以及这种变异如何随输入生物量变化，对于指导对当前文献的有意义解释和规划未来研究至关重要。数据来自2.5年时间内19次单独靶向16 S rRNA基因测序运行的469个测序文库。在去除从阴性对照中鉴定的污染物序列后，244份样品保留了足够的读数用于进一步分析。重复测量的细菌模拟社区内和试验间变异的变异系数范围为8.7%至37.6%（内）和15.6%至80.5%（间）的所有，但一个属的细菌，其相对丰度大于1%。单个粪便样本的试验内与试验间Bray-Curtis成对距离分别为0.11与0.31，而来自同一供体的重复粪便样本的试验内变异更大，为0.38（Wilcoxon p = 0.001）。使用细菌模拟群落的稀释系列来评估输入生物量对可变性的影响。成对的距离增加与稀释样品，和相对丰度的估计变得不可靠，低于每微升约100个拷贝的16 S rRNA基因。使用这些数据，我们创建了一个预测模型，以估计给定输入生物量和相对丰度值的微生物组测量的预期变化。控制良好的微生物组研究足够稳健，可以捕获小的生物学效应，并可以实现与临床测定一致的变异性水平。相对丰度是负相关的变异性的措施，并有一个更强的影响比绝对生物量的变异性，这表明它是可行的，以检测非常低的生物量样品中的细菌种群的差异。此外，通过量化生物量和相对丰度对组成变异性的影响，我们开发了一种工具，用于定义给定微生物组研究中的预期方差。本文的在线版本（10.1186/s40168-018-0543-z）包含补充材料，可供授权用户使用。
Recent advances in sequencing technologies and bioinformatics tools have allowed for large-scale microbiome studies that are rapidly advancing medical research. However, small changes in technique or analysis can significantly alter the results and lead to conflicting findings. Quantifying the technical versus biological variation expected in targeted 16S rRNA gene sequencing studies and how this variation changes with input biomass is critical to guide meaningful interpretation of the current literature and plan future research. Data were compiled from 469 sequencing libraries across 19 separate targeted 16S rRNA gene sequencing runs over a 2.5-year time period. Following removal of contaminant sequences identified from negative controls, 244 samples retained sufficient reads for further analysis. Coefficients of variation for intra- and inter-assay variation from repeated measurements of a bacterial mock community ranged from 8.7 to 37.6% (intra) and 15.6 to 80.5% (inter) for all but one genus of bacteria whose relative abundance was greater than 1%. Intra- versus inter-assay Bray-Curtis pairwise distances for a single stool sample were 0.11 versus 0.31, whereas intra-assay variation from repeat stool samples from the same donor was greater at 0.38 (Wilcoxon p = 0.001). A dilution series of the bacterial mock community was used to assess the effect of input biomass on variability. Pairwise distances increased with more dilute samples, and estimates of relative abundance became unreliable below approximately 100 copies of the 16S rRNA gene per microliter. Using this data, we created a prediction model to estimate the expected variation in microbiome measurements for given input biomass and relative abundance values. Well-controlled microbiome studies are sufficiently robust to capture small biological effects and can achieve levels of variability consistent with clinical assays. Relative abundance is negatively associated with measures of variability and has a stronger effect on variability than does absolute biomass, suggesting that it is feasible to detect differences in bacterial populations in very low-biomass samples. Further, by quantifying the effect of biomass and relative abundance on compositional variability, we developed a tool for defining the expected variance in a given microbiome study. The online version of this article (10.1186/s40168-018-0543-z) contains supplementary material, which is available to authorized users.
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影响因子：
3.7
作者：
Clooney AG;Fouhy F;Sleator RD;O' Driscoll A;Stanton C;Cotter PD;Claesson MJ
通讯作者：
Claesson MJ
影响因子：
15.5
作者：
Kim D;Hofstaedter CE;Zhao C;Mattei L;Tanes C;Clarke E;Lauder A;Sherrill-Mix S;Chehoud C;Kelsen J;Conrad M;Collman RG;Baldassano R;Bushman FD;Bittinger K
通讯作者：
Bittinger K
影响因子：
4.4
作者：
Pollock J;Glendinning L;Wisedchanwet T;Watson M
通讯作者：
Watson M
DOI：
10.1038/ismej.2012.8
发表时间：
2012-08
期刊：
The ISME journal
影响因子：
--
作者：
通讯作者：
--
影响因子：
4.2
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
通讯作者：
Buck GA