Binpairs: Utilization of Illumina Paired-End Information for Improving Efficiency of Taxonomic Binning of Metagenomic Sequences

Motivation

Paired-end sequencing protocols, offered by next generation sequencing (NGS) platforms like Illumia, generate a pair of reads for every DNA fragment in a sample. Although this protocol has been utilized for several metagenomics studies, most taxonomic binning approaches classify each of the reads (forming a pair), independently. The present work explores some simple but effective strategies of utilizing pairing-information of Illumina short reads for improving the accuracy of taxonomic binning of metagenomic datasets. The strategies proposed can be used in conjunction with all genres of existing binning methods.

Results

Validation results suggest that employment of these “Binpairs” strategies can provide significant improvements in the binning outcome. The quality of the taxonomic assignments thus obtained are often comparable to those that can only be achieved with relatively longer reads obtained using other NGS platforms (such as Roche).

Availability

An implementation of the proposed strategies of utilizing pairing information is freely available for academic users at https://metagenomics.atc.tcs.com/binning/binpairs.     

Evidence for mate‐encounter Allee effect in an invasive longhorn beetle (Coleoptera: Cerambycidae)

1. Limited empirical support is available for mate‐encounter Allee effects in invasive insects due to the logistical challenges of studying demographic trends in low‐density populations. 2. Traps baited with pheromone and spruce volatiles were used to monitor the abundance of female Tetropium fuscum F. (Coleoptera: Cerambycidae) at multiple sites in Nova Scotia in 2011 and 2012. Each female was dissected to determine the presence or absence of sperm in its spermatheca (mated or virgin female, respectively). 3. Both male and total T. fuscum abundance declined with increasing distance to the focal point of T. fuscum's invasion. Female mating probability declined with male abundance and with distance from the invasion focus, and mating probabilities were very low at the most peripheral sites. Difficulty in encountering mates may thus contribute to limiting the spread of T. fuscum. 4. The approach outlined here could be integrated into existing surveys of wood borers using traps baited with semiochemicals to improve our understanding of the role of the mate‐encounter Allee effect in invasion dynamics.     

Empirical evaluation of preservation methods for faecal DNA

We evaluate the relative effectiveness of four methods for preserving faecal samples for DNA analysis. PCR assays of fresh faecal samples collected from free-ranging baboons showed that amplification success was dependent on preservation method, PCR-product size, and whether nuclear or mitochondrial DNA was assayed. Storage in a DMSO/EDTA/Tris/salt solution (DETs) was most effective for preserving nuclear DNA, but storage in 70% ethanol, freezing at –20°C and drying performed approximately equally well for mitochondrial DNA and short (<200 bp) nuclear DNA fragments. Because faecal DNA is diluted and degraded, repeated extractions from faeces may be necessary and short nuclear markers should be employed for genotyping. A review of molecular scatology studies further suggests that three to six faeces per individual should be collected.     

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.     

Reversal in competitive dominance of a toxic versus non-toxic cyanobacterium in response to rising CO2

Climate change scenarios predict a doubling of the atmospheric CO₂ concentration by the end of this century. Yet, how rising CO₂ will affect the species composition of aquatic microbial communities is still largely an open question. In this study, we develop a resource competition model to investigate competition for dissolved inorganic carbon in dense algal blooms. The model predicts how dynamic changes in carbon chemistry, pH and light conditions during bloom development feed back on competing phytoplankton species. We test the model predictions in chemostat experiments with monocultures and mixtures of a toxic and non-toxic strain of the freshwater cyanobacterium Microcystis aeruginosa. The toxic strain was able to reduce dissolved CO₂ to lower concentrations than the non-toxic strain, and became dominant in competition at low CO₂ levels. Conversely, the non-toxic strain could grow at lower light levels, and became dominant in competition at high CO₂ levels but low light availability. The model captured the observed reversal in competitive dominance, and was quantitatively in good agreement with the results of the competition experiments. To assess whether microcystins might have a role in this reversal of competitive dominance, we performed further competition experiments with the wild-type strain M. aeruginosa PCC 7806 and its mcyB mutant impaired in microcystin production. The microcystin-producing wild type had a strong selective advantage at low CO₂ levels but not at high CO₂ levels. Our results thus demonstrate both in theory and experiment that rising CO₂ levels can alter the community composition and toxicity of harmful algal blooms.