Bibiana G. Crespo

Taxonomic composition of the particle-attached and free-living bacterial assemblages in the Northwest Mediterranean Sea analyzed by pyrosequencing of the 16S rRNA.

Free‐living (FL) and particle‐attached (PA) bacterial assemblages in the Northwest Mediterranean Sea were studied using pyrosequencing data of the 16S rRNA. We have described and compared the richness, the distribution of Operational Taxonomic Units (OTUs) within the two fractions, the spatial distribution, and the taxonomic composition of FL and PA bacterial assemblages. The number of OTUs in the present work was two orders of magnitude higher than in previous studies. Only 25% of the total OTUs were common to both fractions, whereas 49% OTUs were exclusive to the PA fraction and 26% to the FL fraction. The OTUs exclusively present in PA or FL assemblages were very low in abundance (6% of total abundance). Detection of the rare OTUs revealed the larger richness of PA bacteria that was hidden in previous studies. Alpha‐Proteobacteria dominated the FL bacterial assemblage and gamma‐Proteobacteria dominated the PA fraction. Bacteroidetes were important in the PA fraction mainly at the coast. The high number of sequences in this study detected additional phyla from the PA fraction, such as Actinobacteria, Firmicutes, and Verrucomicrobia.

Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

High-throughput sequencing (HTS) techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample). Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568–1,669 (95% Credible Interval) and 5,027–5,196 for surface and deep water samples respectively, implying that 84–89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

Probing the fabric of the rare biosphere

The relatively recent development of high-throughput sequencing (HTS) techniques has revealed a wealth of novel sequences found in very low abundance: the rare biosphere. We performed a deep (1 million sequences per sample) pyrosequencing analysis of two marine bacterial samples and isolated a culture collection from one of them. Species data were derived from the sequencing analysis (97% similarity criterion) and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total richness of 1 568‒1 669 (95% CI) and 5027‒5196 for surface and deep water samples respectively, implying that 84‒89% of the total richness was sequenced. We also predict that a quadrupling of the present sequencing effort should suffice to observe 90% the total richness in both samples. Comparing with isolate sequences we found that isolation retrieved mainly extremely rare taxa which were not obtained by HTS despite the high sequencing effort. Culturing therefore remains a useful tool for mapping marine bacterial diversity, in addition to its other uses for studying the ecology of the rare biosphere.The relatively recent development of high-throughput sequencing (HTS) techniques has revealed a wealth of novel sequences found in very low abundance: the rare biosphere. Today, most studies of diversity of microorganisms are carried out almost exclusively with HTS techniques. However, culturing seems indispensable for diversity studies especially if the aim is exploring the rare biosphere. We have carried out a deep (1 million sequences per sample) pyrosequencing analysis of two marine bacterial samples and isolated a culture collection from one of them. We have shown that the collectors curves of the pyrosequencing data were close to reaching an asymptote. We have estimated the total richness of the samples and the sequencing effort necessary to obtain the total estimated richness experimentally. Comparing the pyrosequencing data and the isolates sequences we have found that isolation retrieves some of the rarest taxa and that the composition of rare taxa follows an annual succession. We have shown that increasing the number of tags sequenced would slowly uncover the isolated bacteria. However, even if the whole bacterial diversity was found by increasing the sampling depth, culturing would still be essential for the study of marine bacterial communities, especially if the target is the rare biosphere.