Bacterial diversity of metagenomic and PCR libraries from the Delaware River
Bacterial diversity of metagenomic and PCR libraries from the Delaware River
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DOI：
10.1111/j.1462-2920.2005.00762.x
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发表时间：
2005-12-01
影响因子：
5.1
通讯作者：
Kirchman, DL
中科院分区：
文献类型：
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作者：
Cottrell, MT;Waidner, LA;Kirchman, DL
为了确定宏基因组文库是否充分采样了水生环境中的优势细菌，我们研究了用来自特拉华州河的细菌DNA构建的大型插入宏基因组文库的系统发育组成、16 S rRNA基因的聚合酶链反应（PCR）文库以及通过荧光原位杂交（FISH）确定的群落结构。由FISH确定的图书馆和社区结构的组成不同的主要细菌群体在河流中，其中包括放线菌，β-变形菌和噬细胞样细菌。与PCR文库和FISH相比，宏基因组文库中的β-变形菌代表性不足，而噬细胞样细菌在宏基因组文库中比在PCR文库和根据FISH的实际社区中更丰富。特拉华州河文库包含属于几个广泛分布的淡水菌群的细菌，包括多核嗜热菌群、Rhodoferax sp. Bal 47和LD 28 β-变形菌群、放线菌群的ACK-m1和STA 2 -30群以及PRD 01 a001 B类噬细胞菌菌群。对于宏基因组文库和PCR文库，具有> 97%序列同一性的细菌的覆盖率分别为65%和50%。重复PCR文库和用重新调节的扩增子构建的文库的稀疏分析表明，异源双链体形成基本上不影响PCR文库的组成。这项研究表明，虽然它可能会错过一些细菌群体，宏基因组方法可以采样其他群体（例如。G.噬细胞样细菌），这可能是其他培养独立的方法代表性不足。
To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization ( FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups ( e. g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches.