Reconstructing the Genomic Content of Microbiome Taxa through Shotgun Metagenomic Deconvolution

Metagenomics has transformed our understanding of the microbial world, allowing researchers to bypass the need to isolate and culture individual taxa and to directly characterize both the taxonomic and gene compositions of environmental samples. However, associating the genes found in a metagenomic sample with the specific taxa of origin remains a critical challenge. Existing binning methods, based on nucleotide composition or alignment to reference genomes allow only a coarse-grained classification and rely heavily on the availability of sequenced genomes from closely related taxa. Here, we introduce a novel computational framework, integrating variation in gene abundances across multiple samples with taxonomic abundance data to deconvolve metagenomic samples into taxa-specific gene profiles and to reconstruct the genomic content of community members. This assembly-free method is not bounded by various factors limiting previously described methods of metagenomic binning or metagenomic assembly and represents a fundamentally different approach to metagenomic-based genome reconstruction. An implementation of this framework is available at http://elbo.gs.washington.edu/software.html. We first describe the mathematical foundations of our framework and discuss considerations for implementing its various components. We demonstrate the ability of this framework to accurately deconvolve a set of metagenomic samples and to recover the gene content of individual taxa using synthetic metagenomic samples. We specifically characterize determinants of prediction accuracy and examine the impact of annotation errors on the reconstructed genomes. We finally apply metagenomic deconvolution to samples from the Human Microbiome Project, successfully reconstructing genus-level genomic content of various microbial genera, based solely on variation in gene count. These reconstructed genera are shown to correctly capture genus-specific properties. With the accumulation of metagenomic data, this deconvolution framework provides an essential tool for characterizing microbial taxa never before seen, laying the foundation for addressing fundamental questions concerning the taxa comprising diverse microbial communities.     

Basin‐scale phenology and effects of climate variability on global timing of initial seaward migration of Atlantic salmon (Salmo salar)

Migrations between different habitats are key events in the lives of many organisms. Such movements involve annually recurring travel over long distances usually triggered by seasonal changes in the environment. Often, the migration is associated with travel to or from reproduction areas to regions of growth. Young anadromous Atlantic salmon (Salmo salar) emigrate from freshwater nursery areas during spring and early summer to feed and grow in the North Atlantic Ocean. The transition from the freshwater (‘parr’) stage to the migratory stage where they descend streams and enter salt water (‘smolt’) is characterized by morphological, physiological and behavioural changes where the timing of this parr‐smolt transition is cued by photoperiod and water temperature. Environmental conditions in the freshwater habitat control the downstream migration and contribute to within‐ and among‐river variation in migratory timing. Moreover, the timing of the freshwater emigration has likely evolved to meet environmental conditions in the ocean as these affect growth and survival of the post‐smolts. Using generalized additive mixed‐effects modelling, we analysed spatio‐temporal variations in the dates of downstream smolt migration in 67 rivers throughout the North Atlantic during the last five decades and found that migrations were earlier in populations in the east than the west. After accounting for this spatial effect, the initiation of the downstream migration among rivers was positively associated with freshwater temperatures, up to about 10 °C and levelling off at higher values, and with sea‐surface temperatures. Earlier migration occurred when river discharge levels were low but increasing. On average, the initiation of the smolt seaward migration has occurred 2.5 days earlier per decade throughout the basin of the North Atlantic. This shift in phenology matches changes in air, river, and ocean temperatures, suggesting that Atlantic salmon emigration is responding to the current global climate changes.     

Development and refinement of pregnane X receptor (PXR) DNA binding site model using information theory: insights into PXR-mediated gene regulation

The pregnane X receptor (PXR) acts as a receptor to induce gene expression in response to structurally diverse xenobiotics through binding as a heterodimer with the 9-cis retinoic acid receptor (RXR) to enhancers in target gene promoters. We identified and estimated the affinities of novel PXR/RXR binding sites in regulated genes and additional genomic targets of PXR with an information theory-based model of the PXR/RXR binding site. Our initial PXR/RXR model, the result of the alignment of 15 previously characterized binding sites, was used to scan the promoters of known PXR target genes. Sites from these genes, with information contents of >8 bits bound by PXR/RXR in vitro, were used to revise the information weight matrix; this procedure was repeated by screening for progressively weaker binding sites. After three iterations of refinement, the model was based on 48 validated PXR/RXR binding sites and has an average information content (Rsequence) of 14.43 +/- 3.21 bits. A scan of the human genome predicted novel PXR/RXR binding sites in the promoters of UGT1A3 (19.78 bits at -8040 and 16.37 bits at -6930) and UGT1A6 (12.74 bits at -9216), both of which were identified previously as targets for PXR. These sites were subsequently demonstrated to specifically bind PXR/RXR in competition electrophoretic mobility shift assays. A strong PXR site was also predicted upstream of the CASP10 gene (18.69 bits at -7872) and was validated by binding studies and reporter assays as a PXR responsive element. This suggests that the PXR-mediated response extends beyond genes involved in drug biotransformation and transport.     

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.     

A genomewide catalogue of single nucleotide polymorphisms in white‐beaked and Atlantic white‐sided dolphins

The field of population genetics is rapidly moving into population genomics as the quantity of data generated by high‐throughput sequencing platforms increases. In this study, we used restriction‐site‐associated DNA sequencing (RADSeq) to recover genomewide genotypes from 70 white‐beaked (Lagenorhynchus albirostris) and 43 Atlantic white‐sided dolphins (L. acutus) gathered throughout their north‐east Atlantic distribution range. Both species are at a high risk of being negatively affected by climate change. Here, we provide a resource of 38 240 RAD‐tags and 52 981 nuclear SNPs shared between both species. We have estimated overall higher levels of nucleotide diversity in white‐sided (π = 0.0492 ± 0.0006%) than in white‐beaked dolphins (π = 0.0300 ± 0.0004%). White‐sided dolphins sampled in the Faroe Islands, belonging to two pods (N = 7 and N = 11), showed similar levels of diversity (π = 0.0317 ± 0.0007% and 0.0267 ± 0.0006%, respectively) compared to unrelated individuals of the same species sampled elsewhere (e.g. π = 0.0285 ± 0.0007% for 11 Scottish individuals). No evidence of higher levels of kinship within pods can be derived from our analyses. When identifying the most likely number of genetic clusters among our sample set, we obtained an estimate of two to four clusters, corresponding to both species and possibly, two further clusters within each species. A higher diversity and lower population structuring was encountered in white‐sided dolphins from the north‐east Atlantic, in line with their preference for pelagic waters, as opposed to white‐beaked dolphins that have a more patchy distribution, mainly across continental shelves.     

Mapping of theLyb-4 gene to different chromosomes in DBA/2J and C3H/HeJ mice

Linkage has been established between the Lyb-4 alloantigen locus and the chromosome 4 markersLyb- 2 andMup- 1 using recombinant inbred (RI) strains. Only 2 of 24 BXD RI strains possess recombinant genotypes with respect to the B cell alloantigen lociLyb- 4 andLyb- 2, for an estimated recombination frequency of 0.024 ±0.019. One additional BXD RI strain was a recombinant with respect toLyb- 4 andMup- 1 (major urinary protein locus) for an estimated recombination frequency of 0.039 ± 0.026. These linkages were confirmed and further quantitated in a (C57BL/6J × DBA/2J)F₁ × C57BL/6J backcross population, in which the recombination frequency betweenLyb- 4 andMup- 1 was 0.049 ± 0.019. No recombination between the expression of Lyb-4.1 antigen and the ability of anti-Lyb-4.1 serum to suppress MLC reactivity was found, indicating that the genes controlling the antigenic determinant which is recognized with cytotoxic antibodies in anti-Lyb-4.1 serum is the same as, or is very closely linked to, the gene which is responsible for augmentation of the MLC response. In contrast, no linkage was observed between the gene controlling the Lyb-4.1 determinant andMup- 1 in RI strain and backcross mice derived from the cross of C3H/HeJ and C57BL/6J. Again, there was complete concordance between the serologically recognized determinant and the ability of anti-Lyb-4.1 serum to suppress the MLC response. Absorption of anti-Lyb-4.1 serum with C3H/HeJ, DBA/2J, and C57BL/6J lymphocytes, followed by the cytotoxic assay of the absorbed sera on lymphocytes of each of these three strains showed that serologically the Lyb-4.1 antigenic determinant on DBA/2 mice was indistinguishable from that on C3H/HeJ mice. Thus, both traits appear to be under the control of single genes in both DBA/2J and C3H/HeJ, but the C3H/HeJ gene appears to be nonallelic and unlinked to the DBA/2J gene.Abbreviations used in this paper LAD lymphocyte activating determinants - LPS lipopolysaccharide - MLC mixed lymphocyte culture - RI recombinant inbred     

Courtship Behavior of Zaprionus indianus (Gupta) (Diptera: Drosophilidae) from Populations Colonizing South America

We describe for the first time the sexual behavior and the courtship song of males of the African fly Zaprionus indianus (Gupta), a recent invader of South America. The male courtship song is formed by monocyclic pulses and the courtship behavior is simple when compared to that of species of Drosophila. Two interpulse interval (IPI) distributions were observed: pre-mounting and mounting. No significant difference was observed between the pre-mounting IPIs of males that descended from three geographical populations from South America. We also observed the songs produced by females and the homosexual behavior exhibited by males. A sequence of bursts is produced by females as a refusal signal against males, while males emit a characteristic song that identifies sex genus, which differs from the courtship song. The short courtship and mating latencies recorded reveal vigorous males and receptive females, respectively.     

Hydration in purple membrane as a function of relative humidity

Neutron diffraction experiments on the purple membrane of Halobacterium halobium as a function of H₂OD₂O exchange in a wide relative humidity range, are described.Increasing relative humidity leads primarily to hydration of the lipid area in the membrane. The exchanged H density is higher in the centre of the protein than at the protein-lipid interface, in support of the hypothesis that the molecule has a hydrophilic interior. However, there is no aqueous pocket in the protein.