Readsynth: short-read simulation for consideration of composition-biases in reduced metagenome sequencing approaches.

BACKGROUND: The application of reduced metagenomic sequencing approaches holds promise as a middle ground between targeted amplicon sequencing and whole metagenome sequencing approaches but has not been widely adopted as a technique. A major barrier to adoption is the lack of read simulation software built to handle characteristic features of these novel approaches. Reduced metagenomic sequencing (RMS) produces unique patterns of fragmentation per genome that are sensitive to restriction enzyme choice, and the non-uniform size selection of these fragments may introduce novel challenges to taxonomic assignment as well as relative abundance estimates. RESULTS: Through the development and application of simulation software, readsynth, we compare simulated metagenomic sequencing libraries with existing RMS data to assess the influence of multiple library preparation and sequencing steps on downstream analytical results. Based on read depth per position, readsynth achieved 0.79 Pearson's correlation and 0.94 Spearman's correlation to these benchmarks. Application of a novel estimation approach, fixed length taxonomic ratios, improved quantification accuracy of simulated human gut microbial communities when compared to estimates of mean or median coverage. CONCLUSIONS: We investigate the possible strengths and weaknesses of applying the RMS technique to profiling microbial communities via simulations with readsynth. The choice of restriction enzymes and size selection steps in library prep are non-trivial decisions that bias downstream profiling and quantification. The simulations investigated in this study illustrate the possible limits of preparing metagenomic libraries with a reduced representation sequencing approach, but also allow for the development of strategies for producing and handling the sequence data produced by this promising application.

ClustFinder: A tool for threshold-delineated clustering of microbial isolates by pairwise genomic distance.

This paper presents ClustFinder, a command line tool designed to automate clustering of genomes based on genomic distance. This tool will aid researchers and public health professionals in the identification of epidemiological clusters. Here, we demonstrate the usage of ClustFinder with example datasets. ClustFinder is available at github.com/Denes-Lab/ClustFinder.

Microsatellite Loci Reveal Genetic Diversity of Asian Callery Pear (Pyrus calleryana) in the Species Native Range and in the North American Cultivars.

Pyrus calleryana Decne. (Callery pear) includes cultivars that in the United States are popular ornamentals in commercial and residential landscapes. Last few decades, this species has increasingly naturalized across portions of the eastern and southern US. However, the mechanisms behind this plant's spread are not well understood. The genetic relationship of present-day P.calleryana trees with their Asian P. calleryana forebears (native trees from China, Japan, and Korea) and the original specimens of US cultivars are unknown. We developed and used 18 microsatellite markers to analyze 147 Pyrus source samples and to articulate the status of genetic diversity within Asian P. calleryana and US cultivars. We hypothesized that Asian P. calleryana specimens and US cultivars would be genetically diverse and would show genetic relatedness. Our data revealed high genetic diversity, high gene flow, and presence of population structure in P. calleryana, potentially relating to the highly invasive capability of this species. Strong evidence for genetic relatedness between Asian P. calleryana specimens and US cultivars was also demonstrated. Our data suggest the source for P. calleryana that have become naturalized in US was China. These results will help understand the genetic complexity of invasive P. calleryana when developing management for escaped populations: In follow-up studies, we use the gSSRs developed here to analyze P. calleryana escape populations from across US.

ngsComposer: an automated pipeline for empirically based NGS data quality filtering.

Next-generation sequencing (NGS) enables massively parallel acquisition of large-scale omics data; however, objective data quality filtering parameters are lacking. Although a useful metric, evidence reveals that platform-generated Phred values overestimate per-base quality scores. We have developed novel and empirically based algorithms that streamline NGS data quality filtering. The pipeline leverages known sequence motifs to enable empirical estimation of error rates, detection of erroneous base calls and removal of contaminating adapter sequence. The performance of motif-based error detection and quality filtering were further validated with read compression rates as an unbiased metric. Elevated error rates at read ends, where known motifs lie, tracked with propagation of erroneous base calls. Barcode swapping, an inherent problem with pooled libraries, was also effectively mitigated. The ngsComposer pipeline is suitable for various NGS protocols and platforms due to the universal concepts on which the algorithms are based.

Intergenerational response to the endocrine disruptor vinclozolin is influenced by maternal genotype and crossing scheme.

In utero exposure to vinclozolin (VIN), an antiandrogenic fungicide, is linked to multigenerational phenotypic and epigenetic effects. Mechanisms remain unclear. We assessed the role of antiandrogenic activity and DNA sequence context by comparing effects of VIN vs. M2 (metabolite with greater antiandrogenic activity) and wild-type C57BL/6 (B6) mice vs. mice carrying mutations at the previously reported VIN-responsive H19/Igf2 locus. First generation offspring from VIN-treated 8nrCG mutant dams exhibited increased body weight and decreased sperm ICR methylation. Second generation pups sired by affected males exhibited decreased neonatal body weight but only when dam was unexposed. Offspring from M2 treatments, B6 dams, 8nrCG sires or additional mutant lines were not similarly affected. Therefore, pup response to VIN over two generations detected here was an 8nrCG-specific maternal effect, independent of antiandrogenic activity. These findings demonstrate that maternal effects and crossing scheme play a major role in multigenerational response to in utero exposures.

A New Metazoan Recombination Rate Record and Consistently High Recombination Rates in the Honey Bee Genus Apis Accompanied by Frequent Inversions but Not Translocations.

Western honey bees (Apis mellifera) far exceed the commonly observed 1­2 meiotic recombination events per chromosome and exhibit the highest Metazoan recombination rate (20 cM/Mb) described thus far. However, the reasons for this exceptional rate of recombination are not sufficiently understood. In a comparative study, we report on the newly constructed genomic linkage maps of Apis florea and Apis dorsata that represent the two honey bee lineages without recombination rate estimates so far. Each linkage map was generated de novo, based on SNP genotypes of haploid male offspring of a single female. The A. florea map spans 4,782 cM with 1,279 markers in 16 linkage groups. The A. dorsata map is 5,762 cM long and contains 1,189 markers in 16 linkage groups. Respectively, these map sizes result in average recombination rate estimates of 20.8 and 25.1 cM/Mb. Synteny analyses indicate that frequent intra-chromosomal rearrangements but no translocations among chromosomes accompany the high rates of recombination during the independent evolution of the three major honey bee lineages. Our results imply a common cause for the evolution of very high recombination rates in Apis. Our findings also suggest that frequent homologous recombination during meiosis might increase ectopic recombination and rearrangements within but not between chromosomes. It remains to be investigated whether the resulting inversions may have been important in the evolutionary differentiation between honey bee species.

Immunogene and viral transcript dynamics during parasitic Varroa destructor mite infection of developing honey bee (Apis mellifera) pupae.

The ectoparasitic Varroa destructor mite is a major contributor to the ongoing honey bee health crisis. Varroa interacts with honey bee viruses, exacerbating their pathogenicity. In addition to vectoring viruses, immunosuppression of the developing honey bee hosts by Varroa has been proposed to explain the synergy between viruses and mites. However, the evidence for honey bee immune suppression by V. destructor is contentious. We systematically studied the quantitative effects of experimentally introduced V. destructor mites on immune gene expression at five specific time points during the development of the honey bee hosts. Mites reproduced normally and were associated with increased titers of deformed wing virus in the developing bees. Our data on different immune genes show little evidence for immunosuppression of honey bees by V. destructor. Experimental wounding of developing bees increases relative immune gene expression and deformed wing virus titers. Combined, these results suggest that mite feeding activity itself and not immunosuppression may contribute to the synergy between viruses and mites. However, our results also suggest that increased expression of honey bee immune genes decreases mite reproductive success, which may be explored to enhance mite control strategies. Finally, our expression data for multiple immune genes across developmental time and different experimental treatments indicates co-regulation of several of these genes and thus improves our understanding of the understudied honey bee immune system.

Reproduction, social behavior, and aging trajectories in honeybee workers.

While a negative correlation between reproduction and life span is commonly observed, specialized reproductive individuals outlive their non-reproductive nestmates in all eusocial species, including the honeybee, Apis mellifera (L). The consequences of reproduction for individual life expectancy can be studied directly by comparing reproductive and non-reproductive workers. We quantified the life span consequences of reproduction in honeybee workers by removal of the queen to trigger worker reproduction. Furthermore, we observed the social behavior of large cohorts of workers under experimental and control conditions to test for associations with individual life expectancy. Worker life expectancy was moderately increased by queen removal. Queenless colonies contained a few long-lived workers, and oviposition behavior was associated with a strong reduction in mortality risk, indicating that a reproductive role confers a significant survival advantage. This finding is further substantiated by an association between brood care behavior and worker longevity that depends on the social environment. In contrast, other in-hive activities, such as fanning, trophallaxis, and allogrooming did not consistently affect worker life expectancy. The influence of foraging varied among replicates. An earlier age of transitioning from in-hive tasks to outside foraging was always associated with shorter life spans, in accordance with previous studies. In sum, our studies quantify how individual mortality is affected by particular social roles and colony environments and demonstrate interactions between the two. The exceptional, positive association between reproduction and longevity in honeybees extends to within-caste plasticity, which may be exploited for mechanistic studies.

Cross-species correlation between queen mating numbers and worker ovary sizes suggests kin conflict may influence ovary size evolution in honeybees.

During social evolution, the ovary size of reproductively specialized honey bee queens has dramatically increased while their workers have evolved much smaller ovaries. However, worker division of labor and reproductive competition under queenless conditions are influenced by worker ovary size. Little comparative information on ovary size exists in the different honey bee species. Here, we report ovariole numbers of freshly dissected workers from six Apis species from two locations in Southeast Asia. The average number of worker ovarioles differs significantly among species. It is strongly correlated with the average mating number of queens, irrespective of body size. Apis dorsata, in particular, is characterized by numerous matings and very large worker ovaries. The relation between queen mating number and ovary size across the six species suggests that individual selection via reproductive competition plays a role in worker ovary size evolution. This indicates that genetic diversity, generated by multiple mating, may bear a fitness cost at the colony level.