PoolParty2: An integrated pipeline for analysing pooled or indexed low-coverage whole-genome sequencing data to discover the genetic basis of diversity.

Whole-genome sequencing data allow survey of variation from across the genome, reducing the constraint of balancing genome sub-sampling with estimating recombination rates and linkage between sampled markers and target loci. As sequencing costs decrease, low-coverage whole-genome sequencing of pooled or indexed-individual samples is commonly utilized to identify loci associated with phenotypes or environmental axes in non-model organisms. There are, however, relatively few publicly available bioinformatic pipelines designed explicitly to analyse these types of data, and fewer still that process the raw sequencing data, provide useful metrics of quality control and then execute analyses. Here, we present an updated version of a bioinformatics pipeline called PoolParty2 that can effectively handle either pooled or indexed DNA samples and includes new features to improve computational efficiency. Using simulated data, we demonstrate the ability of our pipeline to recover segregating variants, estimate their allele frequencies accurately, and identify genomic regions harbouring loci under selection. Based on the simulated data set, we benchmark the efficacy of our pipeline with another bioinformatic suite, angsd, and illustrate the compatibility and complementarity of these suites using angsd to generate genotype likelihoods as input for identifying linkage outlier regions using alignment files and variants provided by PoolParty2. Finally, we apply our updated pipeline to an empirical dataset of low-coverage whole genomic data from population samples of Columbia River steelhead trout (Oncorhynchus mykiss), results from which demonstrate the genomic impacts of decades of artificial selection in a prominent hatchery stock. Thus, we not only demonstrate the utility of PoolParty2 for genomic studies that combine sequencing data from multiple individuals, but also illustrate how it compliments other bioinformatics resources such as angsd.

Genomic surveillance identifies potential risk factors for SARS-CoV-2 transmission at a mid-sized university in a small rural town.

Understanding transmission dynamics of SARS-CoV-2 in institutions of higher education (IHEs) is important because these settings have potential for rapid viral spread. Here, we used genomic surveillance to retrospectively investigate transmission dynamics throughout the 2020-2021 academic year for the University of Idaho ("University"), a mid-sized IHE in a small rural town. We generated genome assemblies for 1168 SARS-CoV-2 samples collected during the academic year, representing 46.8% of positive samples collected from the University population and 49.8% of positive samples collected from the surrounding community ("Community") at the local hospital during this time. Transmission dynamics differed for the University when compared to the Community, with more infection waves that lasted shorter lengths of time, potentially resulting from high-transmission congregate settings along with mitigation efforts implemented by the University to combat outbreaks. We found evidence for low transmission rates between the University and Community, with approximately 8% of transmissions into the Community originating from the University, and approximately 6% of transmissions into the University originating from the Community. Potential transmission risk factors identified for the University included congregate settings such as sorority and fraternity events and residences, holiday travel, and high caseloads in the surrounding community. Knowledge of these risk factors can help the University and other IHEs develop effective mitigation measures for SARS-CoV-2 and similar pathogens.

High-Resolution Taxonomic Characterization Reveals Novel Human Microbial Strains with Potential as Risk Factors and Probiotics for Prediabetes and Type 2 Diabetes.

Alterations in the composition of the gut microbiota is thought to play a key role in causing type 2 diabetes, yet is not fully understood, especially at the strain level. Here, we used long-read DNA sequencing technology of 16S-ITS-23S rRNA genes for high-resolution characterization of gut microbiota in the development of type 2 diabetes. Gut microbiota composition was characterized from fecal DNA from 47 participants divided into 4 cohorts based on glycemic control: normal glycemic control (healthy; n = 21), reversed prediabetes (prediabetes/healthy; n = 8), prediabetes (n = 8), or type 2 diabetes (n = 10). A total of 46 taxa were found to be possibly related to progression from healthy state to type 2 diabetes. Bacteroides coprophilus DSM 18228, Bifidobacterium pseudocatenulatum DSM 20438, and Bifidobacterium adolescentis ATCC 15703 could confer resistance to glucose intolerance. On the other hand, Odoribacter laneus YIT 12061 may be pathogenic as it was found to be more abundant in type 2 diabetes participants than other cohorts. This research increases our understanding of the structural modulation of gut microbiota in the pathogenesis of type 2 diabetes and highlights gut microbiota strains, with the potential for targeted opportunistic pathogen control or consideration for probiotic prophylaxis and treatment.

Whole genome resequencing identifies local adaptation associated with environmental variation for redband trout.

Aquatic ectotherms are predicted to harbour genomic signals of local adaptation resulting from selective pressures driven by the strong influence of climate conditions on body temperature. We investigated local adaptation in redband trout (Oncorhynchus mykiss gairdneri) using genome scans for 547 samples from 11 populations across a wide range of habitats and thermal gradients in the interior Columbia River. We estimated allele frequencies for millions of single nucleotide polymorphism loci (SNPs) across populations using low-coverage whole genome resequencing, and used population structure outlier analyses to identify genomic regions under divergent selection between populations. Twelve genomic regions showed signatures of local adaptation, including two regions associated with genes known to influence migration and developmental timing in salmonids (GREB1L, ROCK1, SIX6). Genotype-environment association analyses indicated that diurnal temperature variation was a strong driver of local adaptation, with signatures of selection driven primarily by divergence of two populations in the northern extreme of the subspecies range. We also found evidence for adaptive differences between high-elevation desert vs. montane habitats at a smaller geographical scale. Finally, we estimated vulnerability of redband trout to future climate change using ecological niche modelling and genetic offset analyses under two climate change scenarios. These analyses predicted substantial habitat loss and strong genetic shifts necessary for adaptation to future habitats, with the greatest vulnerability predicted for high-elevation desert populations. Our results provide new insight into the complexity of local adaptation in salmonids, and important predictions regarding future responses of redband trout to climate change.

The role of neutral and adaptive genomic variation in population diversification and speciation in two ground squirrel species of conservation concern.

Understanding the neutral (demographic) and adaptive processes leading to the differentiation of species and populations is a critical component of evolutionary and conservation biology. In this context, recently diverged taxa represent a unique opportunity to study the process of genetic differentiation. Northern and southern Idaho ground squirrels (Urocitellus brunneus-NIDGS, and U. endemicus-SIDGS, respectively) are a recently diverged pair of sister species that have undergone dramatic declines in the last 50 years and are currently found in metapopulations across restricted spatial areas with distinct environmental pressures. Here we genotyped single-nucleotide polymorphisms (SNPs) from buccal swabs with restriction site-associated DNA sequencing (RADseq). With these data we evaluated neutral genetic structure at both the inter- and intraspecific level, and identified putatively adaptive SNPs using population structure outlier detection and genotype-environment association (GEA) analyses. At the interspecific level, we detected a clear separation between NIDGS and SIDGS, and evidence for adaptive differentiation putatively linked to torpor patterns. At the intraspecific level, we found evidence of both neutral and adaptive differentiation. For NIDGS, elevation appears to be the main driver of adaptive differentiation, while neutral variation patterns match and expand information on the low connectivity between some populations identified in previous studies using microsatellite markers. For SIDGS, neutral substructure generally reflected natural geographical barriers, while adaptive variation reflected differences in land cover and temperature, as well as elevation. These results clearly highlight the roles of neutral and adaptive processes for understanding the complexity of the processes leading to species and population differentiation, which can have important conservation implications in susceptible and threatened species.

A new mouse SNP genotyping assay for speed congenics: combining flexibility, affordability, and power.

BACKGROUND: Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. "Next generation" high throughput DNA sequencing technologies have the potential to decrease cost and increase flexibility and power of speed congenics, but thus far have not been utilized for this purpose. RESULTS: We took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. The assay surveys 1640 genome-wide SNPs known to be polymorphic across > 100 mouse strains, with an expected average of 549 ± 136 SD diagnostic SNPs between each pair of strains. We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain (807-819 SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J (123-139 SNPs). Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample; this information can be used to guide the selection of individuals for the next backcross, and to assess whether individuals have become congenic. We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/IL13 into C57BL/6J; after six generations of backcrosses, offspring were up to 99.8% congenic. CONCLUSIONS: The SNP genotyping assay and bioinformatic pipeline developed here present a valuable tool for increasing the power and decreasing the cost of many studies that depend on speed congenics. The assay is highly flexible and can be used for combinations of strains that are commonly used for speed congenics. The assay could also be used for other techniques including QTL mapping, standard F2 crosses, ancestry analysis, and forensics.

Genomic association with pathogen carriage in bighorn sheep (Ovis canadensis).

Genetic composition can influence host susceptibility to, and transmission of, pathogens, with potential population-level consequences. In bighorn sheep (Ovis canadensis), pneumonia epidemics caused by Mycoplasma ovipneumoniae have been associated with severe population declines and limited recovery across North America. Adult survivors either clear the infection or act as carriers that continually shed M. ovipneumoniae and expose their susceptible offspring, resulting in high rates of lamb mortality for years following the outbreak event. Here, we investigated the influence of genomic composition on persistent carriage of M. ovipneumoniae in a well-studied bighorn sheep herd in the Wallowa Mountains of Oregon, USA. Using 10,605 SNPs generated using RADseq technology for 25 female bighorn sheep, we assessed genomic diversity metrics and employed family-based genome-wide association methodologies to understand variant association and genetic architecture underlying chronic carriage. We observed no differences among genome-wide diversity metrics (heterozygosity and allelic richness) between groups. However, we identified two variant loci of interest and seven associated candidate genes, which may influence carriage status. Further, we found that the SNP panel explained ~55% of the phenotypic variance (SNP-based heritability) for M. ovipneumoniae carriage, though there was considerable uncertainty in these estimates. While small sample sizes limit conclusions drawn here, our study represents one of the first to assess the genomic factors influencing chronic carriage of a pathogen in a wild population and lays a foundation for understanding genomic influence on pathogen persistence in bighorn sheep and other wildlife populations. Future research should incorporate additional individuals as well as distinct herds to further explore the genomic basis of chronic carriage.

Etelis boweni sp. nov., a new cryptic deepwater eteline snapper from the Indo-Pacific (Perciformes: Lutjanidae).

A new species of Etelis is described based on 16 specimens collected from the Red Sea and Western Australia, with confirmed genetic records throughout the Indo-West Pacific. It is similar to and was often misidentified as Etelis carbunculus Cuvier, with both species sharing the diagnostic character of low number of developed gill rakers. Nonetheless, the two species are genetically divergent and differ morphologically in adult body length; proportions of eye, snout, cheek and caudal fin; shape of head, opercular spine and sagittal otolith; and coloration of the tip of the upper caudal fin. Etelis boweni has a wide Indo-west Pacific distribution that largely overlaps with E. carbunculus, and the two species are often caught on the same fishing line.

Genomic signatures of divergent selection are associated with social behaviour for spinner dolphin ecotypes.

Understanding the genomic basis of adaptation is critical for understanding evolutionary processes and predicting how species will respond to environmental change. Spinner dolphins in the eastern tropical Pacific (ETP) present a unique system for studying adaptation. Within this large geographical region are four spinner dolphin ecotypes with weak neutral genetic divergence and no obvious barriers to gene flow, but strong spatial variation in morphology, behaviour and habitat. These ecotypes have large population sizes, which could reduce the effects of drift and facilitate selection. To identify genomic regions putatively under divergent selective pressures between ecotypes, we used genome scans with 8994 RADseq single nucleotide polymorphisms (SNPs) to identify population differentiation outliers and genotype-environment association outliers. Gene ontology enrichment analyses indicated that outlier SNPs from both types of analyses were associated with multiple genes involved in social behaviour and hippocampus development, including 15 genes associated with the human social disorder autism. Evidence for divergent selection on social behaviour is supported by previous evidence that these spinner dolphin ecotypes differ in mating systems and associated social behaviours. In particular, three of the ETP ecotypes probably have a polygynous mating system characterized by strong premating competition among males, whereas the fourth ecotype probably has a polygynandrous mating system characterized by strong postmating competition such as sperm competition. Our results provide evidence that selection for social behaviour may be an evolutionary force driving diversification of spinner dolphins in the ETP, potentially as a result of divergent sexual selection associated with different mating systems. Future studies should further investigate the potential adaptive role of the candidate genes identified here, and could probably find further signatures of selection using whole genome sequence data.

Building a global genomics observatory: Using GEOME (the Genomic Observatories Metadatabase) to expedite and improve deposition and retrieval of genetic data and metadata for biodiversity research.

Genetic data represent a relatively new frontier for our understanding of global biodiversity. Ideally, such data should include both organismal DNA-based genotypes and the ecological context where the organisms were sampled. Yet most tools and standards for data deposition focus exclusively either on genetic or ecological attributes. The Genomic Observatories Metadatabase (GEOME: geome-db.org) provides an intuitive solution for maintaining links between genetic data sets stored by the International Nucleotide Sequence Database Collaboration (INSDC) and their associated ecological metadata. GEOME facilitates the deposition of raw genetic data to INSDCs sequence read archive (SRA) while maintaining persistent links to standards-compliant ecological metadata held in the GEOME database. This approach facilitates findable, accessible, interoperable and reusable data archival practices. Moreover, GEOME enables data management solutions for large collaborative groups and expedites batch retrieval of genetic data from the SRA. The article that follows describes how GEOME can enable genuinely open data workflows for researchers in the field of molecular ecology.

Wireworm (Coleoptera: Elateridae) genomic analysis reveals putative cryptic species, population structure, and adaptation to pest control.

The larvae of click beetles (Coleoptera: Elateridae), known as "wireworms," are agricultural pests that pose a substantial economic threat worldwide. We produced one of the first wireworm genome assemblies (Limonius californicus), and investigated population structure and phylogenetic relationships of three species (L. californicus, L. infuscatus, L. canus) across the northwest US and southwest Canada using genome-wide markers (RADseq) and genome skimming. We found two species (L. californicus and L. infuscatus) are comprised of multiple genetically distinct groups that diverged in the Pleistocene but have no known distinguishing morphological characters, and therefore could be considered cryptic species complexes. We also found within-species population structure across relatively short geographic distances. Genome scans for selection provided preliminary evidence for signatures of adaptation associated with different pesticide treatments in an agricultural field trial for L. canus. We demonstrate that genomic tools can be a strong asset in developing effective wireworm control strategies.

Diet of a rare herbivore based on DNA metabarcoding of feces: Selection, seasonality, and survival.

In herbivores, survival and reproduction are influenced by quality and quantity of forage, and hence, diet and foraging behavior are the foundation of an herbivore's life history strategy. Given the importance of diet to most herbivores, it is imperative that we know the species of plants they prefer, especially for herbivorous species that are at risk for extinction. However, it is often difficult to identify the diet of small herbivores because: (a) They are difficult to observe, (b) collecting stomach contents requires sacrificing animals, and (c) microhistology requires accurately identifying taxa from partially digested plant fragments and likely overemphasizes less-digestible taxa. The northern Idaho ground squirrel (Urocitellus brunneus) is federally threatened in the United States under the Endangered Species Act. We used DNA metabarcoding techniques to identify the diet of 188 squirrels at 11 study sites from fecal samples. We identified 42 families, 126 genera, and 120 species of plants in the squirrel's diet. Our use of three gene regions was beneficial because reliance on only one gene region (e.g., only trnL) would have caused us to miss >30% of the taxa in their diet. Northern Idaho ground squirrel diet differed between spring and summer, frequency of many plants in the diet differed from their frequency within their foraging areas (evidence of selective foraging), and several plant genera in their diet were associated with survival. Our results suggest that while these squirrels are generalists (they consume a wide variety of plant species), they are also selective and do not eat plants relative to availability. Consumption of particular genera such as Perideridia may be associated with higher overwinter survival.

Range-Wide Population Structure of 3 Deepwater Eteline Snappers Across the Indo-Pacific Basin.

Deep-sea habitats may drive unique dispersal and demographic patterns for fishes, but population genetic analyses to address these questions have rarely been conducted for fishes in these environments. This study investigates the population structure of 3 tropical deepwater snappers of the genus Etelis that reside at 100-400 m depth, with broad and overlapping distributions in the Indo-Pacific. Previous studies showed little population structure within the Hawaiian Archipelago for 2 of these species: Etelis coruscans and E. carbunculus. Here we extend sampling to the entire geographic range of each species to resolve the population genetic architecture for these 2 species, as well as a recently exposed cryptic species (Etelis sp.). One goal was to determine whether deepwater snappers are more dispersive than shallow-water fishes. A second goal was to determine whether submesophotic fishes have older, more stable populations than shallow reef denizens that are subject to glacial sea-level fluctuations. Both goals are pertinent to the management of these valuable food fishes. A total of 1153 specimens of E. coruscans from 15 geographic regions were analyzed, along with 1064 specimens of E. carbunculus from 11 regions, and 590 specimens of E. sp. from 16 regions. The first 2 species were analyzed with mtDNA and 9-11 microsatellite loci, while E. sp. was analyzed with mtDNA only. Etelis coruscans had a non-significant microsatellite global FST, but significant global mtDNA Ф ST = 0.010 (P = 0.0007), with the isolation of Seychelles in the western Indian Ocean, and intermittent signals of isolation for the Hawaiian Archipelago. Etelis carbunculus had a non-significant microsatellite global FST, and significant global mtDNA Ф ST = 0.021 (P = 0.0001), with low but significant levels of isolation for Hawai'i, and divergence between Tonga and Fiji. Etelis sp. had mtDNA Ф ST = 0.018 (P = 0.0005), with a strong pattern of isolation for both Seychelles and Tonga. Overall, we observed low population structure, shallow mtDNA coalescence (similar to near-shore species), and isolation at the fringes of the Indo-Pacific basin in Hawai'i and the western Indian Ocean. While most shallow-water species have population structure on the scale of biogeographic provinces, deepwater snapper populations are structured on the wider scale of ocean basins, more similar to pelagic fishes than to shallow-water species. This population structure indicates the capacity for widespread dispersal throughout the Indo-Pacific region.

Long-lived marine species may be resilient to environmental variability through a temporal portfolio effect.

Maintenance of genetic variation may provide resilience of populations to natural environmental variability. We used Pacific ocean perch (POP; Sebastes alutus) to test for the maintenance of adaptive variation across overlapping generations. POP are a long-lived species characterized by widespread larval dispersal in their first year and a longevity of over 100 years. In order to understand how early marine dispersal affects POP survival and population structure, we used restriction site-associated DNA sequencing (RADseq) to obtain 11,146 single-nucleotide polymorphisms (SNPs) from 401 young-of-the-year (YOY) POP collected during surveys conducted in 2014 (19 stations) and 2015 (4 stations) in the eastern Gulf of Alaska. Population clustering analysis showed that the POP samples represented four distinct ancestral populations mixed throughout the sampling area. Based on prior work on larval dispersal of POP, these larvae are most likely from distinct parturition locations that are mixing during their pelagic dispersal life stage. Latent factor mixed models revealed that POP larvae face significant selection during their first year at sea, which is specific to the year of their birth. Thus each adult cohort's genetic composition is heavily influenced by the environmental conditions experienced during their first year at sea. Long-lived species relying on broadcast spawning strategies may therefore be uniquely resilient to environmental variability by maintaining a portfolio of cohort-specific adaptive genotypes, and age truncation due to overfishing of older cohorts may have detrimental effect on the population viability.

Phylogenomics of the genus Tursiops and closely related Delphininae reveals extensive reticulation among lineages and provides inference about eco-evolutionary drivers.

Phylogeographic inference has provided extensive insight into the relative roles of geographical isolation and ecological processes during evolutionary radiations. However, the importance of cross-lineage admixture in facilitating adaptive radiations is increasingly being recognised, and suggested as a main cause of phylogenetic uncertainty. In this study, we used a double digest RADseq protocol to provide a high resolution (~4 Million bp) nuclear phylogeny of the Delphininae. Phylogenetic resolution of this group has been especially intractable, likely because it has experienced a recent species radiation. We carried out cross-lineage reticulation analyses, and tested for several sources of potential bias in determining phylogenies from genome sampling data. We assessed the divergence time and historical demography of T. truncatus and T. aduncus by sequencing the T. aduncus genome and comparing it with the T. truncatus reference genome. Our results suggest monophyly for the genus Tursiops, with the recently proposed T. australis species falling within the T. aduncus lineage. We also show the presence of extensive cross-lineage gene flow between pelagic and European coastal ecotypes of T. truncatus, as well as in the early stages of diversification between spotted (Stenella frontalis; Stenella attenuata), spinner (Stenella longirostris), striped (Stenella coeruleoalba), common (Delphinus delphis), and Fraser's (Lagenodelphis hosei) dolphins. Our study suggests that cross-lineage gene flow in this group has been more extensive and complex than previously thought. In the context of biogeography and local habitat dependence, these results improve our understanding of the evolutionary processes determining the history of this lineage.

A bioinformatic pipeline for identifying informative SNP panels for parentage assignment from RADseq data.

The development of high-throughput sequencing technologies is dramatically increasing the use of single nucleotide polymorphisms (SNPs) across the field of genetics, but most parentage studies of wild populations still rely on microsatellites. We developed a bioinformatic pipeline for identifying SNP panels that are informative for parentage analysis from restriction site-associated DNA sequencing (RADseq) data. This pipeline includes options for analysis with or without a reference genome, and provides methods to maximize genotyping accuracy and select sets of unlinked loci that have high statistical power. We test this pipeline on small populations of Mexican gray wolf and bighorn sheep, for which parentage analyses are expected to be challenging due to low genetic diversity and the presence of many closely related individuals. We compare the results of parentage analysis across SNP panels generated with or without the use of a reference genome, and between SNPs and microsatellites. For Mexican gray wolf, we conducted parentage analyses for 30 pups from a single cohort where samples were available from 64% of possible mothers and 53% of possible fathers, and the accuracy of parentage assignments could be estimated because true identities of parents were known a priori based on field data. For bighorn sheep, we conducted maternity analyses for 39 lambs from five cohorts where 77% of possible mothers were sampled, but true identities of parents were unknown. Analyses with and without a reference genome produced SNP panels with ≥95% parentage assignment accuracy for Mexican gray wolf, outperforming microsatellites at 78% accuracy. Maternity assignments were completely consistent across all SNP panels for the bighorn sheep, and were 74.4% consistent with assignments from microsatellites. Accuracy and consistency of parentage analysis were not reduced when using as few as 284 SNPs for Mexican gray wolf and 142 SNPs for bighorn sheep, indicating our pipeline can be used to develop SNP genotyping assays for parentage analysis with relatively small numbers of loci.

Age-specific infectious period shapes dynamics of pneumonia in bighorn sheep.

Superspreading, the phenomenon where a small proportion of individuals contribute disproportionately to new infections, has profound effects on disease dynamics. Superspreading can arise through variation in contacts, infectiousness or infectious periods. The latter has received little attention, yet it drives the dynamics of many diseases of critical public health, livestock health and conservation concern. Here, we present rare evidence of variation in infectious periods underlying a superspreading phenomenon in a free-ranging wildlife system. We detected persistent infections of Mycoplasma ovipneumoniae, the primary causative agent of pneumonia in bighorn sheep (Ovis canadensis), in a small number of older individuals that were homozygous at an immunologically relevant genetic locus. Interactions among age-structure, genetic composition and infectious periods may drive feedbacks in disease dynamics that determine the magnitude of population response to infection. Accordingly, variation in initial conditions may explain divergent population responses to infection that range from recovery to catastrophic decline and extirpation.

Comment on "Whole-genome sequence analysis shows two endemic species of North American wolf are admixtures of the coyote and gray wolf".

Whole-genome data do not support a recent hybrid origin for red and eastern wolves.