deep sequencing analyses of low density microbial communities working at the boundary of accurate microbiota detection深度测序分析低密度微生物群落在准确的微生物群的边界检测工作.pdf

Deep Sequencing Analyses of Low Density Microbial Communities: Working at the Boundary of Accurate Microbiota Detection 1 1 2 1 2 Giske Biesbroek , Elisabeth A. M. Sanders , Guus Roeselers , Xinhui Wang , Martien P. M. Caspers , ´ 1 1 . 2. Krzysztof Trzcinski , Debby Bogaert * , Bart J. F. Keijser 1 Department of Pediatric Infectious Diseases and Immunity UMC Utrecht, Utrecht, The Netherlands, 2 Research Group Microbiology and Systems Biology, TNO Earth, Environmental and Life Sciences, Zeist, The Netherlands Abstract 3 4 Introduction: Accurate analyses of microbiota composition of low-density communities (10 –10 bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. This study was aiming to establish protocols for accurate microbiota analysis at low microbial density. Methods: To examine possible effects of bacterial density on microbiota analyses we compared microbiota profiles of serial diluted saliva and low (nares, nasopharynx) and high-density (oropharynx) upper airway communities in four healthy individuals. DNA was extracted with four different extraction methods (Epicentre Masterpure, Qiagen DNeasy, Mobio Powersoil and a phenol bead-beating protocol combined with Agowa-Mag-mini). Bacterial DNA recovery was analysed by 16S qPCR and microbiota profiles through GS-FLX-Titanium-Sequencing of 16S rRNA gene amplicons s