Bacterial diversity of metagenomic and PCR libraries from the Delaware River

Abstract

Summary To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environ- ments, 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- proteobac- teria and Cytophaga -like bacteria. Beta- proteobacte- ria 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 Dela- ware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius , Rhodof- erax 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 repli- cate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composi- tion 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.