Trophic resource partitioning drives fine‐scale coexistence in cryptic bat species

Understanding the processes that enable species coexistence has important implications for assessing how ecological systems will respond to global change. Morphology and functional similarity increase the potential for competition, and therefore, co‐occurring morphologically similar but genetically unique species are a good model system for testing coexistence mechanisms. We used DNA metabarcoding and high‐throughput sequencing to characterize for the first time the trophic ecology of two recently described cryptic bat species with parapatric ranges, Myotis escalerai and Myotis crypticus. We collected fecal samples from allopatric and sympatric regions and from syntopic and allotopic locations within the sympatric region to describe the diets both taxonomically and functionally and compare prey consumption with prey availability. The two bat species had highly similar diets characterized by high arthropod diversity, particularly Lepidoptera, Diptera and Araneae, and a high proportion of prey that is not volant at night, which points to extensive use of gleaning. Diet overlap at the prey item level was lower in syntopic populations, supporting trophic shift under fine‐scale co‐occurrence. Furthermore, the diet of M. escalerai had a marginally lower proportion of not nocturnally volant prey in syntopic populations, suggesting that the shift in diet may be driven by a change in foraging mode. Our findings suggest that fine‐scale coexistence mechanisms can have implications for maintaining broad‐scale diversity patterns. This study highlights the importance of including both allopatric and sympatric populations and choosing meaningful spatial scales for detecting ecological patterns. We conclude that a combination of high taxonomic resolution with a functional approach helps identify patterns of niche shift.     

Evolutionary insight from whole-genome sequencing of experimentally evolved microbes

Experimental evolution (EE) combined with whole‐genome sequencing (WGS) has become a compelling approach to study the fundamental mechanisms and processes that drive evolution. Most EE‐WGS studies published to date have used microbes, owing to their ease of propagation and manipulation in the laboratory and relatively small genome sizes. These experiments are particularly suited to answer long‐standing questions such as: How many mutations underlie adaptive evolution, and how are they distributed across the genome and through time? Are there general rules or principles governing which genes contribute to adaptation, and are certain kinds of genes more likely to be targets than others? How common is epistasis among adaptive mutations, and what does this reveal about the variety of genetic routes to adaptation? How common is parallel evolution, where the same mutations evolve repeatedly and independently in response to similar selective pressures? Here, we summarize the significant findings of this body of work, identify important emerging trends and propose promising directions for future research. We also outline an example of a computational pipeline for use in EE‐WGS studies, based on freely available bioinformatics tools.     

Comparison of the experimental methods in haplotype sequencing via next generation sequencing

Although the diploid nature has been observed for over 50 years, phasing the diploid is still a laborious task. The speed and throughput of next generation sequencing have largely increased in the past decades. However, the short read-length remains one of the biggest challenges of haplotype analysis. For instance, reads as short as 150 bp span no more than one variant in most cases. Numerous experimental technologies have been developed to overcome this challenge. Distance, complexity and accuracy of the linkages obtained are the main factors to evaluate the efficiency of whole genome haplotyping methods. Here, we review these experimental technologies, evaluating their efficiency in linkages obtaining and system complexity. The technologies are organized into four categories based on its strategy: (i) chromosomes separation, (ii) dilution pools, (iii) crosslinking and proximity ligation, (ix) long-read technologies. Within each category, several subsections are listed to classify each technology. Innovative experimental strategies are expected to have high-quality performance, low cost and be labor-saving, which will be largely desired in the future.     

Elucidating cryptic dynamics of Theileria communities in African buffalo using a high‐throughput sequencing informatics approach

1. Increasing access to next‐generation sequencing (NGS) technologies is revolutionizing the life sciences. In disease ecology, NGS‐based methods have the potential to provide higher‐resolution data on communities of parasites found in individual hosts as well as host populations.
2. Here, we demonstrate how a novel analytical method, utilizing high‐throughput sequencing of PCR amplicons, can be used to explore variation in blood‐borne parasite (Theileria—Apicomplexa: Piroplasmida) communities of African buffalo at higher resolutions than has been obtained with conventional molecular tools.
3. Results reveal temporal patterns of synchronized and opposite fluctuations of prevalence and relative abundance of Theileria spp. within the host population, suggesting heterogeneous transmission across taxa. Furthermore, we show that the community composition of Theileria spp. and their subtypes varies considerably between buffalo, with differences in composition reflected in mean and variance of overall parasitemia, thereby showing potential to elucidate previously unexplained contrasts in infection outcomes for host individuals.
4. Importantly, our methods are generalizable as they can be utilized to describe blood‐borne parasite communities in any host species. Furthermore, our methodological framework can be adapted to any parasite system given the appropriate genetic marker.
5. The findings of this study demonstrate how a novel NGS‐based analytical approach can provide fine‐scale, quantitative data, unlocking opportunities for discovery in disease ecology.

     

Discovering genetic polymorphisms in next-generation sequencing data

The ongoing revolution in DNA sequencing technology now enables the reading of thousands of millions of nucleotide bases in a single instrument run. However, this data quantity is often compromised by poor confidence in the read quality. The identification of genetic polymorphisms from this data is therefore problematic and, combined with the vast quantity of data, poses a major bioinformatics challenge. However, once these difficulties have been addressed, next-generation sequencing will offer a means to identify and characterize the wealth of genetic polymorphisms underlying the vast phenotypic variation in biological systems. We describe the recent advances in next-generation sequencing technology, together with preliminary approaches that can be applied for single nucleotide polymorphism discovery in plant species.     

Applications of sequencing technology in clinical microbial infection

Infectious diseases are a type of disease caused by pathogenic microorganisms. Although the discovery of antibiotics changed the treatment of infectious diseases and reduced the mortality of bacterial infections, resistant bacterial strains have emerged. Anti‐infective therapy based on aetiological evidence is the gold standard for clinical treatment, but the time lag and low positive culture rate of traditional methods of pathogen diagnosis leads to relative difficulty in obtaining the evidence of pathogens. Compared with traditional methods of pathogenic diagnosis, next‐generation and third‐generation sequencing technologies have many advantages in the detection of pathogenic microorganisms. In this review, we mainly introduce recent progress in research on pathogenic diagnostic technology and the applications of sequencing technology in the diagnosis of pathogenic microorganisms. This review provides new insights into the application of sequencing technology in the clinical diagnosis of microorganisms.     

Habitat selection as a mechanism of resource partitioning in two cryptic bat species Pipistrellus pipistrellus and Pipistrellus pygmaeus

Ecomorphological studies of bat communities often reveal the spatial and temporal coexistence of morphologically similar species, leading to suggestions that these communities are structured by non-deterministic processes. However, the diversification of echolocation call structure in bats allows for considerable morphological similarity while still permitting niche differentiation based on specialisation for prey type and habitat structure. The recent separation of a common Palaearctic bat, the pipistrelle, into Pipistrellus pipistrellus and P. pygmaeus, which are sympatrically distributed throughout their range, raises the question as to whether these two morphologically similar species partition resources in time and space.
To test the hypothesis that the coexistence of these cryptic species is facilitated by differential habitat use, 14 P . pipistrellus , and 12 P. pygmaeus were radio-tracked from adjacent maternity roosts, in northeast Scotland, from May to September 2002/2003. The two species showed distinct habitat partitioning with P. pygmaeus foraging predominantly in riparian woodland and over water, and P. pipistrellus foraging along woodland edges and short isolated tree lines. Inter-specific overlap in habitat use was low and consequently foraging ranges were segregated spatially.
The degree of habitat partitioning revealed in these species, which show considerable overlap in echolocation call parameters and functional morphology, suggests that morphological features, whilst useful in separating chiropteran species into coarse-grained foraging guilds, may not predict fine-grained ecological segregation.     

Perspectives of DNA microarray and next-generation DNA sequencing technologies

DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research. Supported by the National High-Tech Research Program of China (Grant No.2006AA020704) and Shanghai Science and Technology Commission (Grant No. 05DZ22201)     

Deep sequencing identified potential miRNAs involved in defence response,stress and plant growth characteristics of wild genotypes of cardamom

• Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
• In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
• We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
• Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.

     

Coexisting cryptic species of the Litoditis marina complex (Nematoda) show differential resource use and have distinct microbiomes with high intraspecific variability

Differences in resource use or in tolerances to abiotic conditions are often invoked as potential mechanisms underlying the sympatric distribution of cryptic species. Additionally, the microbiome can provide physiological adaptations of the host to environmental conditions. We determined the intra‐ and interspecific variability of the microbiomes of three cryptic nematode species of the Litoditis marina species complex that co‐occur, but show differences in abiotic tolerances. Roche 454 pyrosequencing of the microbial 16S rRNA gene revealed distinct bacterial communities characterized by a substantial diversity (85–513 OTUs) and many rare OTUs. The core microbiome of each species contained only very few OTUs (2–6), and four OTUs were identified as potentially generating tolerance to abiotic conditions. A controlled experiment in which nematodes from two cryptic species (Pm1 and Pm3) were fed with either an E. coli suspension or a bacterial mix was performed, and the 16S rRNA gene was sequenced using the MiSeq technology. OTU richness was 10‐fold higher compared to the 454 data set and ranged between 1118 and 7864. This experiment confirmed the existence of species‐specific microbiomes, a core microbiome with few OTUs, and high interindividual variability. The offered food source affected the bacterial community and illustrated different feeding behaviour between the cryptic species, with Pm3 exhibiting a higher degree of selective feeding than Pm1. Morphologically similar species belonging to the same feeding guild (bacterivores) can thus have substantial differences in their associated microbiomes and feeding strategy, which in turn may have important ramifications for biodiversity–ecosystem functioning relationships.     

Looking for therapeutic antibodies in next-generation sequencing repositories

ABSTRACT

Recently it has become possible to query the great diversity of natural antibody repertoires using next-generation sequencing (NGS). These methods are capable of producing millions of sequences in a single experiment. Here we compare clinical-stage therapeutic antibodies to the ~1b sequences from 60 independent sequencing studies in the Observed Antibody Space database, which includes antibody sequences from NGS analysis of immunoglobulin gene repertoires. Of 242 post-Phase 1 antibodies, we found 16 with sequence identity matches of 95% or better for both heavy and light chains. There are also 54 perfect matches to therapeutic CDR-H3 regions in the NGS outputs, suggesting a nontrivial amount of convergence between naturally observed sequences and those developed artificially. This has potential implications for both the legal protection of commercial antibodies and the discovery of antibody therapeutics.     

金针菇基因组测序与组装策略分析

采用二代和三代测序技术分别对金针菇单核体菌株“6-3”进行测序,应用4种组装策略进行基因组的de novo组装,对比组装效果。基因组组装的参数方面,仅使用二代测序组装的效果最差,长度大于10kb的Contig全长只有24.6Mb,Contig N50只有23kb,组装率只有59.27%。采用三代组装二代校正的组装策略效果最好,长度大于10kb的Contig全长为38.3Mb,Contig N50为2.8Mb,组装率高达92.16%。保守单拷贝基因拼接效果方面,4种组装策略获得基因组序列与BUSCO数据库里的担子菌的保守单拷贝基因比对,基因完整性均大于94%。在组装准确性方面,经过PCR扩增、Sanger测序验证,三代组装二代校正的基因组序列完整并且连续,同时序列上碱基的SNP、InDel数量最少。综上所述,三代组装二代校正得到的基因组序列具有Contig N50值大、组装率高、碱基准确性高的特点,是食用菌基因组测序较为理想的方案。     

Massive parallel sequencing in animal genetics: wherefroms and wheretos

Next generation sequencing (NGS) has revolutionized genomics research, making it difficult to overstate its impact on studies of Biology. NGS will immediately allow researchers working in non‐mainstream species to obtain complete genomes together with a comprehensive catalogue of variants. In addition, RNA‐seq will be a decisive way to annotate genes that cannot be predicted purely by computational or comparative approaches. Future applications include whole genome sequence association studies, as opposed to classical SNP‐based association, and implementing this new source of information into breeding programmes. For these purposes, one of the main advantages of sequencing vs. genotyping is the possibility of identifying copy number variants. Currently, experimental design is a topic of utmost interest, and here we discuss some of the options available, including pools and reduced representation libraries. Although bioinformatics is still an important bottleneck, this limitation is only transient and should not deter animal geneticists from embracing these technologies.     

Sexual niche partitioning in two species of New Guinean Pachycephala whistlers

Sex differences in foraging behavior have been widely reported in the ornithological literature, but few examples are available from tropical avifaunas. Differences between males and females in foraging behavior have been hypothesized to be a byproduct of sexual size dimorphism or a result of niche partitioning to reduce intersexual competition for food or different reproductive roles. From 2010 to 2013, I used foraging data and mist‐net capture rates from multiple study sites to examine possible sex differences in the foraging behavior of two New Guinean Pachycephala whistlers. I found that male Regent (Pachycephala schlegelii) and Sclater's (Pachycephala soror) whistlers consistently foraged in higher strata than females. It is unlikely that these differences are due to sexual dimorphism because these species exhibit little sexual dimorphism. Sex differences in foraging behavior were consistent across years and study sites and did not appear linked to breeding behavior, supporting the food‐competition hypothesis, but not the reproductive‐roles hypothesis. Male territorial defense often occurs in relatively high strata in Pachycephala whistlers, possibly influencing male foraging strata. However, male territorial behavior cannot explain why females predominately forage in lower strata. Instead, intersexual competition for food resources is likely the primary driver of differences in the foraging behavior of male and female Regent and Sclater's whistlers.     

Efficient and sensitive identification and quantification of airborne pollen using next‐generation DNA sequencing

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen‐allergic patients. Current pollen monitoring methods are microscope‐based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost‐effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next‐generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.     

Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex

Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome‐wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next‐generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high‐quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome‐wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi‐species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon‐based PTS to rapidly generate large‐scale data for population genetic and phylogenetic‐based research.     

Milk microbiome signatures of subclinical mastitis-affected cattle analysed by shotgun sequencing

Aims: Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was carried out by next‐generation sequencing 454 GS‐FLX technology to elucidate the microbial community structure of cattle milk. Methods and Results: Milk samples from Kankrej, Gir and crossbred cattle were subjected to metagenomic profiling by pyrosequencing. The Metagenomic analysis produced 63·07, 11·09 and 7·87 million base pairs (Mb) of sequence data, assembled in 264 798, 56 114 and 36 762 sequences with an average read length of 238, 197 and 214 nucleotides in Kankrej, Gir and crossbred cattle, respectively. Phylogenetic and metabolic profiles by the web‐based tool MG‐RAST revealed that the members of Enterobacteriales were predominant in mastitic milk followed by Pseudomonadales, Bacillales and Lactobacillales. Around 56 different species with varying abundance were detected in the subclinically infected milk. Escherichia coli was found to be the most predominant species in Kankrej and Gir cattle followed by Pseudomonas aeruginosa, Pseudomonas mendocina, Shigella flexneri and Bacillus cereus. In crossbred cattle, Staphylococcus aureus followed by Klebsiella pneumoniae, Staphylococcus epidermidis and E. coli were detected in descending order. Metabolic profiling indicated fluoroquinolones, methicillin, copper, cobalt–zinc–cadmium as the groups of antibiotics and toxic compounds to which the organisms showed resistance. Sequences indicating potential of organisms exhibiting multidrug resistance against antibiotics and resistance to toxic compounds were also present. Interestingly, presence of bacteriophages against Staph. aureus, E. coli, Enterobacter and Yersinia species was also observed. Conclusions: The analysis identified potential infectious organisms in mastitis, resistance of organisms to antibiotics and chemical compounds and the natural resistance potential of dairy cows. Significance and Impact of the Study: The findings of this study may help in formulating strategies for the prevention and treatment of mastitis in dairy animals and consequently in reducing economic losses incurred because of it.