Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses.
Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses.
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DOI：
10.1093/nar/gks002
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发表时间：
2012-05
影响因子：
14.9
通讯作者：
Creer S
中科院分区：
文献类型：
--
作者：
Fonseca VG;Nichols B;Lallias D;Quince C;Carvalho GR;Power DM;Creer S
环境样品中的真核生物多样性通常通过基于PCR的nSSU基因扩增来评估。然而，来自焦磷酸测序环境数据集的多样性估计往往被夸大，主要是因为在PCR扩增过程中嵌合序列的形成。嵌合体是由不同的亲本序列组成的杂交产物，这可能导致对多样性估计的误解。我们分析了样品的丰富性，均匀性和系统发育多样性的嵌合体的形成上使用的nSSU数据集来自454罗氏焦磷酸测序的复制，大型控制池的密切和遥远的相关线虫模拟社区，已知的基因组内的身份和丰富性。为了进一步研究嵌合分子是如何形成的，在几个个体中分析了nSSU基因二级结构。对于第一次在真核生物中，嵌合体的形成被证明是更高的更丰富和更遗传多样性的样品，从而提供了一个新的视角嵌合体的形成在焦磷酸测序的环境数据集。研究结果有助于更好地了解嵌合体形成的性质和机制，在PCR扩增环境衍生的DNA。此外，考虑到使用扩增子测序的生物多样性分析与用于评估基因组变异的生物多样性分析之间的相似性，我们的研究结果具有潜在的广泛应用，可用于识别同源基因座或多基因家族中的遗传变异。
Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU data set derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals. For the first time in eukaryotes, chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in pyrosequenced environmental data sets. Findings contribute to a better understanding of the nature and mechanisms involved in chimera formation during PCR amplification of environmentally derived DNA. Moreover, given the similarities between biodiversity analyses using amplicon sequencing and those used to assess genomic variation, our findings have potential broad application for identifying genetic variation in homologous loci or multigene families in general.
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影响因子：
16.6
作者：
Fonseca, Vera G.;Carvalho, Gary R.;Sung, Way;Johnson, Harriet F.;Power, Deborah M.;Neill, Simon P.;Packer, Margaret;Blaxter, Mark L.;Lambshead, P. John D.;Thomas, W. Kelley;Creer, Simon
通讯作者：
Creer, Simon
影响因子：
12.3
作者：
Huse SM;Huber JA;Morrison HG;Sogin ML;Welch DM
通讯作者：
Welch DM
影响因子：
5.8
作者：
Huber, T;Faulkner, G;Hugenholtz, P
通讯作者：
Hugenholtz, P
影响因子：
4.4
作者：
Ashelford, KE;Chuzhanova, NA;Weightman, AJ
通讯作者：
Weightman, AJ
影响因子：
4.4
作者：
Ashelford, Kevin E.;Chuzhanova, Nadia A.;Weightman, Andrew J.
通讯作者：
Weightman, Andrew J.