Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA.

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples

Contributions of Translocation to Northern Bobwhite Population Recovery

The National Bobwhite Conservation Initiative (NBCI 2.0) is a range-wide plan for recovering northern bobwhites (Colinus virginianus; hereafter, bobwhite[s]). Using geospatial analysis informed by expertise from practitioners, the plan categorizes landscapes into restoration potential by weighing biological constraints and opportunities such that targeted habitat management will produce bobwhite population growth. A fundamental assumption of the NBCI 2.0 for achieving recovery goals is that bobwhite source populations currently exist on the landscape at densities necessary to (re)colonize newly established or improved habitat. However, we have found that these source populations can be very low or non-existent, especially in northern tiers of the bobwhite distribution. In 1997, we initiated research to evaluate bobwhite population response following translocation using birds from high density populations to newly developed habitats with low bobwhite numbers (ha). We worked collaboratively with the Georgia Department of Natural Resources in 2006 to develop and implement a wild bobwhite translocation policy based on key findings from that research. Since that time 3,866 wild bobwhites have been trapped and translocated from properties in the Albany and Greater Red Hills region of Florida and Georgia to 13 recipient sites in 6 states (AL, GA, MD, NC, NJ, and SC) on 29,780 ha. A typical translocation was conducted for 2 – 3 years in March by capturing, tagging and transporting birds overnight for release at an average rate of 1 bird per 7 ha per property. Prior to translocation, each recipient property underwent extensive habitat restoration and agreed to conduct a monitoring program including spring whistle counts and fall covey counts before, during, and after translocation. Bobwhite populations increased on recipient sites from an average of 0.38 (CI: 0.13 – 0.63) birds per hectare to 2.2 (CI: 1.45 – 2.95) birds per hectare resulting in the establishment of huntable wild bobwhite populations adding approximately 42,714 bobwhites to the landscape. The value of these wild bobwhites was determined to average $736 per translocated bird bringing the total value of birds donated from the Albany and Greater Red Hills region for translocation to$2,844,564. The establishment of population hubs through translocation contributes to population recovery efforts outlined in the NBCI 2.0, especially where source populations are limited

Development and Implementation of a Successful Northern Bobwhite Translocation Program in Georgia

Gamebird translocations have been used for many years to establish or re-establish populations in North America. A long-term severe decline of northern bobwhites (Colinus virginianus) over much of their range has brought this issue to the forefront for this species as well. Field studies in Georgia over the last decade have documented site fidelity, high survival, reproductive success, and population response from bobwhites translocated into large blocks of well-managed habitat. The Georgia Department of Natural Resources/ Wildlife Resources Division developed an official wild quail translocation policy in 2006. Five translocation projects have been permitted and conducted since the policy was established. Extensive large scale habitat modifications were required on the recipient sites while donor sites were required to have existing high density bobwhite populations. These projects have resulted in translocation of . 800 bobwhites and the establishment of 7,480 ha of new wild quail population centers thereby contributing to the National Bobwhite Conservation Initiative (NBCI) recovery goals. Average fall densities on completed projects have increased from , 0.5 birds/ha to . 1.25 birds/ha

The Sexual Proclivities of Northern Bobwhites

Mating systems and mating behavior are historically viewed as static characteristics of populations. ‘Monogamy’, ‘polygamy’, ‘multiple clutch polygamy’, ‘polyandry’, etc. imply most individuals express similar social and genetic mating patterns and characterize the behavior of most or all individuals in a given population. Mating systems of different populations of northern bobwhites (Colinus virginianus) have been described as monogamous, polygamous, multi-clutch polygamous, ambisexually polygamous, and polyandrous in contrast to the expectation that behavior, at the species level, fits defined categories. Prior studies of bobwhite breeding behavior, to arrive at these classifications, were based on observations of social interactions and did not incorporate genetic outcomes. Thus, it is challenging to discern whether social behaviors predict genetic outcomes and whether bobwhite mating behavior varies among populations or if behavior is flexible within populations. We used 3 years of field observations and variation at 14 microsatellite loci of 601 adult and 841 neonatal bobwhites to estimate rates of extra-pair paternity in bobwhite broods, evaluate the utility of social behavior as an indicator of genetic outcomes, and evaluate the fit of bobwhite social and genetic behavior to classic mating system theories. Extra-pair paternity occurred in \u3e50% of bobwhite broods, whereas extra-pair matings resulted in few (x¯= 1.1) extra-pair offspring per nest. Social interactions between female and male bobwhites generally predicted the father of most offspring in a brood, but social interactions did not predict extra-pair matings better than chance. The mating behavior of individual females changed within and between breeding seasons, suggesting individual reproductive decisions of females were flexible. The mating ‘system’ of bobwhites meets neither the predictions nor the assumptions of classic mating theories

Effect of Meso-Mammal Nest Predator Activity on Northern Bobwhite Nest Success

Perceived changes in predator-prey dynamics along with documented declines of northern bobwhite (Colinus virginianus) have created a renewed interest from biologists and managers about the role meso-mammals play in shaping bobwhite population trajectories. As part of a larger effort to understand this predator-prey relationship, we evaluated meso-mammal activity on sites where bobwhite demographic data was simultaneously being collected via radio- telemetry. During 1999–2006 we conducted 66 meso-mammal activity surveys using scent stations on 16 sites in 3 southeastern states (Florida, Georgia, and Alabama). We calculated an index of meso-mammal activity (PI), for each site, as the average number of station visits per night by raccoons (Procyon lotor), Virginia opossums (Didelphis virginiana), nine-banded armadillos (Dasypus novemcinctus), bobcats (Lynx rufus) and foxes (Vulpes vulpes, Urocyon cinereoargenteus). We collected bobwhite reproductive information on 2,940 nests (x̄ = 45/site, SE = 2.98) from a total of 4,379 radiotagged bobwhites (x̄ = 67/site, SE = 3.81). The average PI for all sites combined was 0.13 (range = 0.03-0.38, SE = 0.01). Nesting success on the sites ranged from 0.29 to 0.72 and averaged 0.52 (SE = 0.01). We used a mixed effects logistic regression model including site as a random effect and observed a negative relationship between the PI and nesting success (β = -1.53 ± 0.72 SE). Our results suggest that meso-mammal activity affects bobwhite nest success, thus, predator management may be warranted at times on some sites to maximize productivity. Future research and modeling efforts should explore the impacts of meso-mammal activity on population growth rates and stability, especially in varying spatial contexts

Survival and Reproduction of Parent-Reared Northern Bobwhites

Captive-reared and released game birds typically have low reproductive success in the wild which limits their use for restoration of game bird populations. A fundamental problem with captive-rearing techniques is the absence of a mechanism for imprinting. We developed a parent-rearing technique that facilitates pre- and post-hatch imprinting using parent-reared wild strain northern bobwhite (Colinus virginianus) chicks in outdoor pens. Parent-reared chicks were marked with patagial wing tags and recaptured during October and the following March. We radiomarked juveniles captured in March to monitor survival and reproductive success in two separate studies, one in Georgia, and one in South Carolina, USA. Band-recapture survival estimates of parent-reared chicks from release to the following breeding season in Georgia (2005–2007) averaged 0.12 (range 1⁄4 0.06 to 0.25) and was dependent on release period. Radio- marked, parent-reared bobwhites had lower survival than wild bobwhites and produced 0.3 nests per hen for the breeding season versus 1.0 nests per hen for radio-marked wild resident bobwhites. Nesting success and subsequent chick survival did not differ among groups, but sample sizes were small. Radio-marked, parent-reared hens (n 1⁄4 26) in the South Carolina study (2008–2010) produced 0.67 nests per hen for the breeding season versus 0.62 nests per hen for radio-marked wild resident hens. Nesting success and brood-rearing success of parent-reared hens did not differ from that of wild resident hens and breeding season survival was also similar. Survival and reproduction of parent-reared wild strain bobwhites were greater than previously reported for pen-reared bobwhites and may useful for restoring or enhancing bobwhites populations at the local scale

Impacts of Red Imported Fire Ants on Northern Bobwhite Nest Survival

Invasive species are a large management issue because of extensive environmental and economic damage. The red imported fire ant (RIFA, Solenopsis invicta) is an invasive species of growing concern in wildlife management in the United States because of its increasing distribution, difficulty to suppress, and aggressive predation on native invertebrates, herpetofauna, birds, and small mammals. Managers of the northern bobwhite (Colinus virginianus) have questioned the direct impact of RIFA on bobwhite nest success, particularly the factors that might influence the level of nest predation. We hypothesized that RIFA predation on northern bobwhite would vary by location and correlate with drier weather conditions. To test our hypothesis we analyzed bobwhite nest data gathered from 3 properties in the southeastern United States. The nest data are part of a larger research effort on bobwhites, collected by Tall Timbers Research Station and the Albany Quail Project using radiotelemetry from 1992 to 2015. Over these 23 years we tracked bobwhites and located nests (n = 3,207) daily to determine nest fate (success or failure) based on field sign and evidence attributed to specific causes of mortality. We used the nest-survival model in Program MARK to estimate nest survival. Average nest survival varied annually for the Albany and Tall Timbers study areas, respectively. The overall effect of RIFAs on nest survival was generally small, with an average annual loss in Albany of 5.13% (range = 0–15.59%) and 2.17% in Tall Timbers (range = 0–5.83%), but in some years was as high as 15.6%. Greater losses occurred late in the breeding season when it was typically drier and the loss rate in general was higher on the drier of the 2 study sites. Despite the relatively small direct impact on nesting success demonstrated, indirect effects of RIFAs on bobwhite populations are poorly understood at present. Quantifying the influence of indirect factors, such as soil type and habitat disturbance, on RIFA abundance and the subsequent impact on bobwhites, will provide additional insight to the ecological interaction with a highly pervasive and expanding invasive species and help inform management options

Habitat Selection by Northern Bobwhite Broods in Pine Savanna Ecosystems

Habitat for northern bobwhite (Colinus virginianus) broods is a critical component of bobwhite management. Research within pine (Pinus spp.) savannas has provided contradictory results regarding the value of macro-habitats with studies demonstrating selection for annually-disked fallow fields and others showing avoidance of fields and selection for burned pine savannas. Field establishment (up to 30% of a property) is a published management recommendation for bobwhites in pine savannas but there are significant annual costs with fallow-field management; information on factors that influence habitat selection by broods can improve management recommendations and facilitate weighing costs/benefits. We examined 2nd and 3rd order habitat selection by 466 broods on 3 sites during 1999–2009. All sites had similar macro-habitats (e.g., pine savanna, fallow fields, hardwood drains) but differed in soil characteristics and species composition of ground vegetation. Annually-disked fields were preferred by broods in most years on sites with predominantly grass and hardwood scrub ground vegetation. Rainfall mediated use of hardwood drains and burned upland pine savannas; hardwood drains were used more during droughts whereas burned pine savannas were used more with increased rainfall. Burned upland pine savanna was preferred on higher fertility sites in 9 of 10 years at the 3rd order level, fields were avoided or used according to availability in 8 of 10 years, and drains were avoided. Managers should consider how soil, weather, and vegetation community in pine savannas influences habitat use by bobwhite broods when identifying the value of different macro-habitats. Field establishment may or may not provide brood habitat depending on site

Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.

BACKGROUND: Blood transcriptional signatures are candidates for non-sputum triage or confirmatory tests of tuberculosis. Prospective head-to-head comparisons of their diagnostic accuracy in real-world settings are necessary to assess their clinical use. We aimed to compare the diagnostic accuracy of candidate transcriptional signatures identified by systematic review, in a setting with a high burden of tuberculosis and HIV. METHODS: We did a prospective observational study nested within a diagnostic accuracy study of sputum Xpert MTB/RIF (Xpert) and Xpert MTB/RIF Ultra (Ultra) tests for pulmonary tuberculosis. We recruited consecutive symptomatic adults aged 18 years or older self-presenting to a tuberculosis clinic in Cape Town, South Africa. Participants provided blood for RNA sequencing, and sputum samples for liquid culture and molecular testing using Xpert and Ultra. We assessed the diagnostic accuracy of candidate blood transcriptional signatures for active tuberculosis (including those intended to distinguish active tuberculosis from other diseases) identified by systematic review, compared with culture or Xpert MTB/RIF positivity as the standard reference. In our primary analysis, patients with tuberculosis were defined as those with either a positive liquid culture or Xpert result. Patients with missing blood RNA or sputum results were excluded. Our primary objective was to benchmark the diagnostic accuracy of candidate transcriptional signatures against the WHO target product profile (TPP) for a tuberculosis triage test. FINDINGS: Between Feb 12, 2016, and July 18, 2017, we obtained paired sputum and RNA sequencing data from 181 participants, 54 (30%) of whom had confirmed pulmonary tuberculosis. Of 27 eligible signatures identified by systematic review, four achieved the highest diagnostic accuracy with similar area under the receiver operating characteristic curves (Sweeney3: 90·6% [95% CI 85·6-95·6]; Kaforou25: 86·9% [80·9-92·9]; Roe3: 86·9% [80·3-93·5]; and BATF2: 86·8% [80·6-93·1]), independent of age, sex, HIV status, previous tuberculosis, or sputum smear result. At test thresholds that gave 70% specificity (the minimum WHO TPP specificity for a triage test), these four signatures achieved sensitivities between 83·3% (95% CI 71·3-91·0) and 90·7% (80·1-96·0). No signature met the optimum criteria, of 95% sensitivity and 80% specificity proposed by WHO for a triage test, or the minimum criteria (of 65% sensitivity and 98% specificity) for a confirmatory test, but all four correctly identified Ultra-positive, culture-negative patients. INTERPRETATION: Selected blood transcriptional signatures met the minimum WHO benchmarks for a tuberculosis triage test but not for a confirmatory test. Further development of the signatures is warranted to investigate their possible effects on clinical and health economic outcomes as part of a triage strategy, or when used as add-on confirmatory test in conjunction with the highly sensitive Ultra test for Mycobacterium tuberculosis DNA. FUNDING: Royal Society Newton Advanced Fellowship, Wellcome Trust, National Institute of Health Research, and UK Medical Research Council