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首页> 外文期刊>Applied Microbiology >rpoD Gene Pyrosequencing for the Assessment of Pseudomonas Diversity in a Water Sample from the Woluwe River
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rpoD Gene Pyrosequencing for the Assessment of Pseudomonas Diversity in a Water Sample from the Woluwe River

机译：rpoD基因焦磷酸测序用于评估沃卢韦河水样中的假单胞菌多样性

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A water sample from a noncontaminated site at the source of the Woluwe River (Belgium) was analyzed by culture-dependent and -independent methods. Pseudomonas isolates were identified by sequencing and analysis of the rpoD gene. Culture-independent methods consisted of cloning and pyrosequencing of a Pseudomonas rpoD amplicon from total DNA extracted from the same sample and amplified with selective rpoD gene primers. Among a total of 14,540 reads, 6,228 corresponded to Pseudomonas rpoD gene sequences by a BLAST analysis in the NCBI database. The selection criteria for the reads were sequences longer than 400 bp, an average Q _(40) value greater than 25, and >85% identity with a Pseudomonas species. Of the 6,228 Pseudomonas rpoD sequences, 5,345 sequences met the established criteria for selection. Sequences were clustered by phylogenetic analysis and by use of the QIIME software package. Representative sequences of each cluster were assigned by BLAST analysis to a known Pseudomonas species when the identity with the type strain was greater than or equal to 96%. Twenty-six species distributed among 12 phylogenetic groups or subgroups within the genus were detected by pyrosequencing. Pseudomonas stutzeri , P. moraviensis , and P. simiae were the only cultured species not detected by pyrosequencing. The predominant phylogenetic group within the Pseudomonas genus was the P. fluorescens group, as determined by culture-dependent and -independent analyses. In all analyses, a high number of putative novel phylospecies was found: 10 were identified in the cultured strains and 246 were detected by pyrosequencing, indicating that the diversity of Pseudomonas species has not been fully described.
机译：通过依赖于文化和不依赖文化的方法分析了来自沃卢威河（比利时）源头未受污染场地的水样。通过对rpoD基因的测序和分析鉴定假单胞菌分离物。不依赖培养物的方法包括克隆和焦磷酸测序假单胞菌rpoD扩增子，该扩增子来自同一样品中提取的总DNA，并用选择性rpoD基因引物扩增。通过NCBI数据库中的BLAST分析，在总共14,540个读数中，有6,228个对应于假单胞菌rpoD基因序列。读取的选择标准是：序列长于400 bp，平均Q_（40）值大于25，且与假单胞菌属的一致性> 85％。在6,228个假单胞菌rpoD序列中，有5,345个序列符合确定的选择标准。通过系统发育分析并使用QIIME软件包对序列进行聚类。当与类型菌株的同一性大于或等于96％时，通过BLAST分析将每个簇的代表性序列分配给已知的假单胞菌属。通过焦磷酸测序检测到在该属的12个系统发育组或亚组中分布的26种。 Stutzeri假单胞菌，M。moraviensis和S. simiae是焦磷酸测序未检出的唯一培养物种。假单胞菌属中主要的系统发生组是荧光假单胞菌组，这是通过培养依赖性和非依赖性分析确定的。在所有分析中，发现了大量推定的新物种：在培养菌株中鉴定出10种，通过焦磷酸测序检测到246种，这表明假单胞菌物种的多样性尚未得到充分描述。

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