Effects of graphene oxide on PCR amplification for microbial community survey
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RESEARCH ARTICLE
Open Access
Effects of graphene oxide on PCR amplification for microbial community survey Shuzhen Li1,2, Zhujun Wang1,3, Yuanyuan Wang4, Maoyong Song4, Guangxin Lu5, Ning Dang5, Huaqun Yin6, Yuanyuan Qu2 and Ye Deng1,3*
Abstract Background: Graphene oxide (GO) has been suggested as an efficient assistant additive to eliminate non-specific amplification of the polymerase chain reaction (PCR). Although many studies have focused on exploring its molecular mechanism, the practice of GO on the quantitation of microbial community has not been implemented yet. In this study, GO was added in PCR system to explore the changes on removing typical amplification errors, such as chimera and mismatches on two kinds of mock communities (an evenly mixed and a staggered mock communities) and environmental samples. Results: High-throughput sequencing of bacterial and fungal communities, based on 16S rRNA genes and internal transcribed spacers (ITS) respectively, showed that GO could significantly increase large segmental error (chimeric sequence) in PCR procedure while had no specific effect on point error (mismatched sequence). Besides, GO reduced the α-diversity of community, and changed the composition of fungal community more obviously than bacterial community. Conclusions: Our study provides the first quantitative data on microbial community level to prove the negative effect of GO, and also indicates that there may be a more complex interaction between GO and comprehensive DNA fragments in PCR process. Keywords: Oxidized graphene, Bacteria, Fungi, Chimera, Community composition
Background In recent years, the study of environmental microbiome is undergoing a great revolution by the development of next-generation sequencing approaches and the establishment of robust bioinformatic tools [1, 2]. The sequential steps of conducting a microbiome study have been systematic and comprehensive raised by researchers [3]. * Correspondence: [email protected] 1 CAS Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China 3 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China Full list of author information is available at the end of the article
After preliminary works finished, for example, sample collection and DNA extraction, PCR-based marker gene survey methods are applied. A segment of a conserved sequence such as the 16S ribosomal RNA (rRNA) gene for bacteria, or internal transcribed spacers (ITS) region for fungi, is amplified and sequenced, to quantify and visualize the microbial community composition, distribution and diversity that made up by operational taxonomic units (OTUs). It has been a common consent that PCR has become one of the most ubiquitous and important tools in molecular biology since it was developed in 1985 [4]. However, the amplification efficiency of PCR often decreased with the
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