Search

Marine planktonic archaea are widespread and abundant in deep oceanic waters but, despite their obvious ecological importance, little is known about them. Metagenomic analyses of large genome fragments allow access to both gene content and genome structure from single individuals of these cultivation-reluctant organisms. We present the comparative analysis of 22 archaeal genomic clones containing 16S rRNA genes that were selected from four metagenomic libraries constructed from meso- and bathypelagic plankton of different oceanic regions (South Atlantic, Antarctic Polar Front, Adriatic and Ionian Sea; depths from 500 to 3000 m). We sequenced clones of the divergent archaeal lineages Group 1A (Crenarchaeota) and Group III (Euryarchaeota) as well as clones from the more frequent Group I Crenarchaeota and Group II Euryarchaeota. Whenever possible, we analysed clones that had identical or nearly identical 16S rRNA genes and that were retrieved from distant geographical locations, that is, that defined pan-oceanic operational taxonomic units (OTUs). We detected genes involved in nitrogen fixation in Group 1A Crenarchaeota, and genes involved in carbon fixation pathways and oligopeptide importers in Group I Crenarchaeota, which could confirm the idea that these are mixotrophic. A two-component system resembling that found in ammonia-oxidizing bacteria was found in Group III Euryarchaeota, while genes for anaerobic respiratory chains were detected in Group II Euryarchaeota. Whereas gene sequence conservation was high, and recombination and gene shuffling extensive within and between OTUs in Group I Crenarchaeota, gene sequence conservation was low and global synteny maintained in Group II Euryarchaeota. This implies remarkable differences in genome dynamics in Group I Crenarchaeota and Group II Euryarchaeota with recombination and mutation being, respectively, the dominant genome-shaping forces. These observations, along with variations in GC content, led us to hypothesize that the two groups of organisms have fundamentally different lifestyles.

To quantify and characterize N and C isotopic fractionation effects due to fungal transformation of organic substrates in forest ecosystems, we performed a field study in California and a meta-analysis of three additional studies conducted by others across the Northern Hemisphere. Basidiomycete fungal biomass was consistently enriched for the heavier isotope for C relative to substrate and either enriched or depleted for N relative to atmospheric N. Extent and pattern of fractionation was very variable, but the distinction between ectomycorrhizal and saprotrophic basidiomycetes was strongly supported, particularly when dual isotope analyses were performed. This differentiation, which we call the "EM-SAP Divide" holds for studies within a restricted ecosystem, but becomes less distinct over larger geographical regions, removing the rationale for using direct isotopic values from single specimens as diagnostic of ecophysiological role. For C, the EM-SAP Divide seems to reflect substrate effects, potentially due to differential access to recently synthesized versus recycled organic compounds, rather than distinct physiological pathways. Once substrate and ecophysiological role effects are removed, our meta-analysis suggests the existence of more than one mechanism causing C fractionations in fungi which is found equally in ectomycorrhizal and saprotrophic fungi. Similarly, a multimodal distribution of δ15N values suggests that physiological effects may play a much stronger influence on N natural isotopic distributions in fungi. Our meta-analysis provides a firm statistical base to evaluate fungal ecological statements based on natural isotopic distributions of C and N. We call into question the current practice of using direct isotopic measurements to make statements about trophic relationships of fungi in the absence of other supporting evidence.

Massively parallel sequencing offers the possibility of revolutionizing the study of viral populations by providing ultra deep sequencing (tens to hundreds of thousand fold coverage) of complete viral genomes. However, differentiation of true low frequency variants from sequencing errors remains challenging. We developed a software package, V-Phaser 2, for inferring intrahost diversity within viral populations. This program adds three major new methodologies to the state of the art: a technique to efficiently utilize paired end read data for calling phased variants, a new strategy to represent and infer length polymorphisms, and an in line filter for erroneous calls arising from systematic sequencing artifacts. We have also heavily optimized memory and run time performance. This combination of algorithmic and technical advances allows V-Phaser 2 to fully utilize extremely deep paired end sequencing data (such as generated by Illumina sequencers) to accurately infer low frequency intrahost variants in viral populations in reasonable time on a standard desktop computer. V-Phaser 2 was validated and compared to both QuRe and the original V-Phaser on three datasets obtained from two viral populations: a mixture of eight known strains of West Nile Virus (WNV) sequenced on both 454 Titanium and Illumina MiSeq and a mixture of twenty-four known strains of WNV sequenced only on 454 Titanium. V-Phaser 2 outperformed the other two programs in both sensitivity and specificity while using more than five fold less time and memory. We developed V-Phaser 2, a publicly available software tool (V-Phaser 2 can be accessed via: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/v-phaser-2 and is freely available for academic use) that enables the efficient analysis of ultra-deep sequencing data produced by common next generation sequencing platforms for viral populations.