Metagenomics of the Deep Chlorophyll Maximum
The Metagenome of the deep chlorophyll maximum in the Mediterranean studied by direct and fosmid library 454 pyrosequencing
We have extracted metagenomic DNA from the pico-planktonic 5-0.2 µm size fraction (mostly prokaryotic cells) from a single sample obtained in mid October at 50 m depth and analyzed the DNA from this sample (without cloning or amplification) by 454 pyrosequencing (hereafter referred to as Direct Sequencing,  DS). We also used 454 to sequence ca. 1152 randomly selected fosmid clones from a metagenomic fosmid library constructed from the same DNA sample. Here we compare the output from both methods and the picture of the microbial community that they provide.
The Deep Chlorophyll Maximum (DCM) is a zone of maximal photosynthetic activity, generally located towards the base of the photic zone in lakes and oceans. The DCM  in the Mediterranean is a seasonal phenomenon. The metagenome from a single sample of a mature Mediterranean DCM community has been 454 pyrosequenced both directly and after cloning in fosmids. The comparison between the two approaches  revealed a bias in the fosmid libraries against low GC DNA and specifically against the two most dominant members of the community, Candidatus Pelagibacter and Prochlorococcus marinus subsp. pastoris, thus unexpectedly providing a feasible method to obtain large genomic fragments from other less prevalent members of this community. This study is the first to be carried out at this sequencing depth (ca. 600 Mbp combining direct and fosmid sequencing) at any DCM. Our results indicate a microbial community massively dominated by the high-light adapted P. marinus subsp. pastoris, Synechococcus sp. and the heterotroph Candidatus Pelagibacter. The sequences retrieved were remarkably similar to the existing genome of P. marinus subsp. pastoris with a nucleotide identity over 98%. Besides, we found a large number of cyanophages that could prey on this microbe although sequence conservation was much lower. The high abundance of phage sequences in the cellular size fraction indicated a remarkably high proportion of cells suffering phage lytic attack. In addition, several fosmids clearly belonging to Group II Euryarchaeota were retrieved and recruited many fragments from the total DNA sequencing suggesting that this group might be quite abundant in this habitat.