Long walk to genomics : history and current approaches to genome sequencing and assembly

Genomes represent the starting point of genetic studies. Since the discovery of DNA structure, scientists have devoted great efforts to determine their sequence in an exact way. In this review we provide a comprehensive historical background of the improvements in DNA sequencing technologies that have accompanied the major milestones in genome sequencing and assembly, ranging from early sequencing methods to Next-Generation Sequencing platforms. We then focus on the advantages and challenges of the current technologies and approaches, collectively known as Third Generation Sequencing. As these technical advancements have been accompanied by progress in analytical methods, we also review the bioinformatic tools currently employed in de novo genome assembly, as well as some applications of Third Generation Sequencing technologies and high-quality reference genomes

molecular enrichment for detection of s aureus in recreational waters

The identification of rapid methods for the control of recreational water and of aquatic environments with similar characteristics is necessary to provide adequate levels of health safety for users. Molecular techniques have been proposed in recent years as a viable alternative to traditional microbiological methods, as they offer various advantages and are less time consuming than traditional tests. An innovative protocol based on molecular enrichment that allows the identification of low concentrations of Staphylococcus aureus in recreational water has been developed. The method is based on the specific amplification of prokaryotic genomic DNA by the usage of universal primers for 23S rDNA; subsequently, a second amplification step is performed with specific real-time polymerase chain reaction (PCR) primers and probe. This approach shows sensitivity levels similar to those observed with microbiological tests, with the additional benefits of the specificity typical of nucleic acids techniques. This methodology is easily applicable also to other microbiological parameters, representing an important milestone in hygiene monitoring by the detection of specific pollution indicators

Antimicrobial Effectiveness of Innovative Photocatalysts: A Review

Waterborne pathogens represent one of the most widespread environmental concerns. Conventional disinfection methods, including chlorination and UV, pose several operational and environmental problems; namely, formation of potentially hazardous disinfection by-products (DBPs) and high energy consumption. Therefore, there is high demand for effective, low-cost disinfection treatments. Among advanced oxidation processes, the photocatalytic process, a form of green technology, is becoming increasingly attractive. A systematic review was carried out on the synthesis, characterization, toxicity, and antimicrobial performance of innovative engineered photocatalysts. In recent decades, various engineered photocatalysts have been developed to overcome the limits of conventional photocatalysts using different synthesis methods, and these are discussed together with the main parameters influencing the process behaviors. The potential environmental risks of engineered photocatalysts are also addressed, considering the toxicity effects presented in the literature

SMRT long reads and Direct Label and Stain optical maps allow the generation of a high-quality genome assembly for the European barn swallow (Hirundo rustica rustica)

Background: The barn swallow (Hirundo rustica) is a migratory bird that has been the focus of a large number of ecological, behavioral, and genetic studies. To facilitate further population genetics and genomic studies, we present a reference genome assembly for the European subspecies (H. r. rustica). Findings: As part of the Genome10K effort on generating high-quality vertebrate genomes (Vertebrate Genomes Project), we have assembled a highly contiguous genome assembly using single molecule real-time (SMRT) DNA sequencing and several Bionano optical map technologies. We compared and integrated optical maps derived from both the Nick, Label, Repair, and Stain technology and from the Direct Label and Stain (DLS) technology. As proposed by Bionano, DLS more than doubled the scaffold N50 with respect to the nickase. The dual enzyme hybrid scaffold led to a further marginal increase in scaffold N50 and an overall increase of confidence in the scaffolds. After removal of haplotigs, the final assembly is approximately 1.21 Gbp in size, with a scaffold N50 value of more than 25.95 Mbp. Conclusions: This high-quality genome assembly represents a valuable resource for future studies of population genetics and genomics in the barn swallow and for studies concerning the evolution of avian genomes. It also represents one of the very first genomes assembled by combining SMRT long-read sequencing with the new Bionano DLS technology for scaffolding. The quality of this assembly demonstrates the potential of this methodology to substantially increase the contiguity of genome assemblies

Monitoring COVID-19 Transmission Risks by Quantitative RealTime PCR Tracing of Droplets in Hospital and Living Environments

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) environmental contamination occurs through droplets and biological fluids released in the surroundings from patients or asymptomatic carriers. Surfaces and objects contaminated by saliva or nose secretions represent a risk for indirect transmission of coronavirus disease 2019 (COVID-19). We assayed surfaces from hospital and living spaces to identify the presence of viral RNA and the spread of fomites in the environment. Anthropic contamination by droplets and biological fluids was monitored by detecting the microbiota signature using multiplex quantitative real-time PCR (qPCR) on selected species and massive sequencing on 16S amplicons. A total of 92 samples (flocked swabs) were collected from critical areas during the pandemic, including indoor (three hospitals and three public buildings) and outdoor surfaces exposed to anthropic contamination (handles and handrails, playgrounds). Traces of biological fluids were frequently detected in spaces open to the public and on objects that are touched with the hands (.80%). However, viral RNA was not detected in hospital wards or other indoor and outdoor surfaces either in the air system of a COVID hospital but only in the surroundings of an infected patient, in consistent association with droplet traces and fomites. Handled objects accumulated the highest level of multiple contaminations by saliva, nose secretions, and fecal traces, further supporting the priority role of handwashing in prevention. In conclusion, anthropic contamination by droplets and biological fluids is widespread in spaces open to the public and can be traced by qPCR. Monitoring fomites can support evaluation of indirect transmission risks for coronavirus or other flu-like viruses in the environment

Adcyap1 polymorphism covaries with breeding latitude in a Nearctic migratory songbird, the Wilson's warbler (Cardellina pusilla)

Understanding the genetic background of complex behavioral traits, showing multigenic control and extensive environmental effects, is a challenging task. Among such traits, migration is known to show a large additive genetic component. Yet, the identification of specific genes or gene regions explaining phenotypic variance in migratory behavior has received less attention. Migration ultimately depends on seasonal cycles, and polymorphism at phenological candidate genes may underlie variation in timing of migration or other aspects of migratory behavior. In this study of a Nearctic-Neotropical migratory songbird, the Wilson's warbler (Cardellina pusilla), we investigated the association between polymorphism at two phenological candidate genes, Clock and Adcyap1, and two aspects of the migratory phenotype, timing of spring migration through a stopover site and inferred latitude of the breeding destination. The breeding destination of migrating individuals was identified using feather deuterium ratio (\u3b42H), which reliably reflects breeding latitude throughout the species' western breeding range. Ninety-eight percent of the individuals were homozygous at Clock, and the rare heterozygotes did not deviate from homozygous migration phenology. Adcyap1 was highly polymorphic, and allele size was not significantly associated with migration date. However, Adcyap1 allele size significantly positively predicted the inferred breeding latitude of males but not of females. Moreover, we found a strong positive association between inferred breeding latitude and Adcyap1 allele size in long-distance migrating birds from the northern sector of the breeding range (western Canada), while this was not the case in short-distance migrating birds from the southern sector of the breeding range (coastal California). Our findings support previous evidence for a role of Adcyap1 in shaping the avian migratory phenotype, while highlighting that patterns of phenological candidate gene-phenotype associations may be complex, significantly varying between geographically distinct populations and even between the sexes

The Mitogenome Relationships and Phylogeography of Barn Swallows (Hirundo rustica)

The barn swallow (Hirundo rustica) poses a number of fascinating scientific questions, including the taxonomic status of postulated subspecies. Here, we obtained and assessed the sequence variation of 411 complete mitogenomes, mainly from the European H. r. rustica, but other subspecies as well. In almost every case, we observed subspecies-specific haplogroups, which we employed together with estimated radiation times to postulate a model for the geographical and temporal worldwide spread of the species. The female barn swallow carrying the Hirundo rustica ancestral mitogenome left Africa (or its vicinity) around 280 thousand years ago (kya), and her descendants expanded first into Eurasia and then, at least 51kya, into the Americas, from where a relatively recent (<20kya) back migration to Asia took place. The exception to the haplogroup subspecies specificity is represented by the sedentary Levantine H. r.Transitiva that extensively shares haplogroup A with the migratory European H. r. rustica and, to a lesser extent, haplogroup B with the Egyptian H. r. savignii. Our data indicate that rustica and transitiva most likely derive from a sedentary Levantine population source that split at the end of the Younger Dryas (YD) (11.7kya). Since then, however, transitiva received genetic inputs from and admixed with both the closely related rustica and the adjacent savignii. Demographic analyses confirm this species' strong link with climate fluctuations and human activities making it an excellent indicator for monitoring and assessing the impact of current global changes on wildlife

Inter-generational resemblance of methylation levels at circadian genes and associations with phenology in the barn swallow.

Regulation of gene expression can occur via epigenetic effects as mediated by DNA methylation. The potential for epigenetic effects to be transmitted across generations, thus modulating phenotypic variation and affecting ecological and evolutionary processes, is increasingly appreciated. However, the study of variation in epigenomes and inter-generational transmission of epigenetic alterations in wild populations is at its very infancy. We studied sex- and age-related variation in DNA methylation and parent-offspring resemblance in methylation profiles in the barn swallows. We focused on a class of highly conserved 'clock' genes (clock, cry1, per2, per3, timeless) relevant in the timing of activities of major ecological importance. In addition, we considerably expanded previous analyses on the relationship between methylation at clock genes and breeding date, a key fitness trait in barn swallows. We found positive assortative mating for methylation at one clock locus. Methylation varied between the nestling and the adult stage, and according to sex. Individuals with relatively high methylation as nestlings also had high methylation levels when adults. Extensive parent-nestling resemblance in methylation levels was observed. Occurrence of extra-pair fertilizations allowed to disclose evidence hinting at a prevalence of paternal germline or sperm quality effects over common environment effects in generating father-offspring resemblance in methylation. Finally, we found an association between methylation at the clock poly-Q region, but not at other loci, and breeding date. We thus provided evidence for sex-dependent variation and the first account of parent-offspring resemblance in methylation in any wild vertebrate. We also showed that epigenetics may influence phenotypic plasticity of timing of life cycle events, thus having a major impact on fitness

Clock gene polymorphism and scheduling of migration : a geolocator study of the barn swallow Hirundo rustica

Circannual rhythms often rely on endogenous seasonal photoperiodic timers involving 'clock' genes, and Clock gene polymorphism has been associated to variation in phenology in some bird species. In the long-distance migratory barn swallow Hirundo rustica, individuals bearing the rare Clock allele with the largest number of C-terminal polyglutamine repeats found in this species (Q8) show a delayed reproduction and moult later. We explored the association between Clock polymorphism and migration scheduling, as gauged by light-level geolocators, in two barn swallow populations (Switzerland; Po Plain, Italy). Genetic polymorphism was low: 91% of the 64 individuals tracked year-round were Q7/Q7 homozygotes. We compared the phenology of the rare genotypes with the phenotypic distribution of Q7/Q7 homozygotes within each population. In Switzerland, compared to Q7/Q7, two Q6/Q7 males departed earlier from the wintering grounds and arrived earlier to their colony in spring, while a single Q7/Q8 female was delayed for both phenophases. On the other hand, in the Po Plain, three Q6/Q7 individuals had a similar phenology compared to Q7/Q7. The Swiss data are suggestive for a role of genetic polymorphism at a candidate phenological gene in shaping migration traits, and support the idea that Clock polymorphism underlies phenological variation in birds