Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with “state‐of‐the‐art” equipment and well‐trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore‐concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN‐1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.     

SimRAD: an R package for simulation‐based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches

Application of high‐throughput sequencing platforms in the field of ecology and evolutionary biology is developing quickly with the introduction of efficient methods to reduce genome complexity. Numerous approaches for genome complexity reduction have been developed using different combinations of restriction enzymes, library construction strategies and fragment size selection. As a result, the choice of which techniques to use may become cumbersome, because it is difficult to anticipate the number of loci resulting from each method. We developed SimRAD, an R package that performs in silico restriction enzyme digests and fragment size selection as implemented in most restriction site associated DNA polymorphism and genotyping by sequencing methods. In silico digestion is performed on a reference genome or on a randomly generated DNA sequence when no reference genome sequence is available. SimRAD accurately predicts the number of loci under alternative protocols when a reference genome sequence is available for the targeted species (or a close relative) but may be unreliable when no reference genome is available. SimRAD is also useful for fine‐tuning a given protocol to adjust the number of targeted loci. Here, we outline the functionality of SimRAD and provide an illustrative example of the use of the package (available on the CRAN at http://cran.r-project.org/web/packages/SimRAD ).     

Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR‐based single‐molecule sequencing approach

While standard DNA‐sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time‐consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large‐scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost‐effective manner. As a solution, we developed a simple PCR‐based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water‐in‐oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate.     

Algorithmic single‐locus species delimitation: effects of sampling effort,variation and nonmonophyly in four methods and 1870 species of beetles

The vast number of undescribed species and the fast rate of biodiversity loss call for new approaches to speed up alpha taxonomy. A plethora of methods for delimiting species or operational taxonomic units (OTUs) based on sequence data have been published in recent years. We test the ability of four delimitation methods (BIN, ABGD, GMYC, PTP) to reproduce established species boundaries on a carefully curated DNA barcode data set of 1870 North European beetle species. We also explore how sampling effort, intraspecific variation, nearest neighbour divergence and nonmonophyly affect the OTU delimitations. All methods produced approximately 90% identity between species and OTUs. The effects of variation and sampling differed between methods. ABGD was sensitive to singleton sequences, while GMYC showed tendencies for oversplitting. The best fit between species and OTUs was achieved using simple rules to find consensus between discordant OTU delimitations. Using several approaches simultaneously allows the methods to compensate for each other's weaknesses. Barcode‐based OTU‐picking is an efficient way to delimit putative species from large data sets where the use of more sophisticated methods based on multilocus or genomic data is not feasible.     

Bayesian species identification under the multispecies coalescent provides significant improvements to DNA barcoding analyses

DNA barcoding methods use a single locus (usually the mitochondrial COI gene) to assign unidentified specimens to known species in a library based on a genetic distance threshold that distinguishes between‐species divergence from within‐species diversity. Recently developed species delimitation methods based on the multispecies coalescent (MSC) model offer an alternative approach to individual assignment using either single‐locus or multiloci sequence data. Here, we use simulations to demonstrate three features of an MSC method implemented in the program bpp . First, we show that with one locus, MSC can accurately assign individuals to species without the need for arbitrarily determined distance thresholds (as required for barcoding methods). We provide an example in which no single threshold or barcoding gap exists that can be used to assign all specimens without incurring high error rates. Second, we show that bpp can identify cryptic species that may be misidentified as a single species within the library, potentially improving the accuracy of barcoding libraries. Third, we show that taxon rarity does not present any particular problems for species assignments using bpp and that accurate assignments can be achieved even when only one or a few loci are available. Thus, concerns that have been raised that MSC methods may have problems analysing rare taxa (singletons) are unfounded. Currently, barcoding methods enjoy a huge computational advantage over MSC methods and may be the only approach feasible for massively large data sets, but MSC methods may offer a more stringent test for species that are tentatively assigned by barcoding.     

Tissue storage and primer selection influence pyrosequencing‐based inferences of diversity and community composition of endolichenic and endophytic fungi

Next‐generation sequencing technologies have provided unprecedented insights into fungal diversity and ecology. However, intrinsic biases and insufficient quality control in next‐generation methods can lead to difficult‐to‐detect errors in estimating fungal community richness, distributions and composition. The aim of this study was to examine how tissue storage prior to DNA extraction, primer design and various quality‐control approaches commonly used in 454 amplicon pyrosequencing might influence ecological inferences in studies of endophytic and endolichenic fungi. We first contrast 454 data sets generated contemporaneously from subsets of the same plant and lichen tissues that were stored in CTAB buffer, dried in silica gel or freshly frozen prior to DNA extraction. We show that storage in silica gel markedly limits the recovery of sequence data and yields a small fraction of the diversity observed by the other two methods. Using lichen mycobiont sequences as internal positive controls, we next show that despite careful filtering of raw reads and utilization of current best‐practice OTU clustering methods, homopolymer errors in sequences representing rare taxa artificially increased estimates of richness c. 15‐fold in a model data set. Third, we show that inferences regarding endolichenic diversity can be improved using a novel primer that reduces amplification of the mycobiont. Together, our results provide a rationale for selecting tissue treatment regimes prior to DNA extraction, demonstrate the efficacy of reducing mycobiont amplification in studies of the fungal microbiomes of lichen thalli and highlight the difficulties in differentiating true information about fungal biodiversity from methodological artefacts.     

A total crapshoot? Evaluating bioinformatic decisions in animal diet metabarcoding analyses

Metabarcoding studies provide a powerful approach to estimate the diversity and abundance of organisms in mixed communities in nature. While strategies exist for optimizing sample and sequence library preparation, best practices for bioinformatic processing of amplicon sequence data are lacking in animal diet studies. Here we evaluate how decisions made in core bioinformatic processes, including sequence filtering, database design, and classification, can influence animal metabarcoding results. We show that denoising methods have lower error rates compared to traditional clustering methods, although these differences are largely mitigated by removing low‐abundance sequence variants. We also found that available reference datasets from GenBank and BOLD for the animal marker gene cytochrome oxidase I (COI) can be complementary, and we discuss methods to improve existing databases to include versioned releases. Taxonomic classification methods can dramatically affect results. For example, the commonly used Barcode of Life Database (BOLD) Classification API assigned fewer names to samples from order through species levels using both a mock community and bat guano samples compared to all other classifiers (vsearch‐SINTAX and q2‐feature‐classifier's BLAST + LCA, VSEARCH + LCA, and Naive Bayes classifiers). The lack of consensus on bioinformatics best practices limits comparisons among studies and may introduce biases. Our work suggests that biological mock communities offer a useful standard to evaluate the myriad computational decisions impacting animal metabarcoding accuracy. Further, these comparisons highlight the need for continual evaluations as new tools are adopted to ensure that the inferences drawn reflect meaningful biology instead of digital artifacts.     

Hybrid dynamics in a species group of swallowtail butterflies

Hybrid zones provide unique natural laboratories for studying mechanisms of evolution. But identification and classification of hybrid individuals (F1, F2, backcross, etc.) can be complicated by real population changes over time as well as by use of different marker types, both of which challenge documentation of hybrid dynamics. Here, we use multiple genetic markers (mitochondrial DNA, microsatellites and genomewide single nucleotide polymorphisms) to re‐examine population structure in a hybrid zone between two species of swallowtail butterflies in western Canada, Papilio machaon and P. zelicaon. Our aim was to test whether their hybrid dynamics remain the same as found 30 years ago using morphology and allozymes, and we compared different genetic data sets as well as alternative hybrid identification and classification methods. Overall, we found high differentiation between the two parental species, corroborating previous research from the 1980s. We identified fewer hybrid individuals in the main zone of hybridization in recent years, but this finding depended on the genetic markers considered. Comparison of methods with simulated data sets generated from our data showed that single nucleotide polymorphisms were more powerful than microsatellites for both hybrid identification and classification. Moreover, substantial variation among comparisons underlined the value of multiple markers and methods for documenting evolutionarily dynamic systems.     

Is DNA barcoding child's play? Science education and the utility of DNA barcoding for the discrimination of UK tree species

We present the findings of a DNA barcoding study of the UK tree flora, implemented as part of an innovative, research‐based science education programme called ‘Tree School’. The UK tree flora comprises native and introduced species, and is a taxonomically diverse study group for the exploration of the potential and limitations of DNA barcoding. The children participating in the project collected voucher specimens and generated DNA barcode sequences from trees and shrubs found in the grounds and surrounding woodlands of a residential field centre in Dorset, UK. We assessed the potential of rbcL and matK markers for amplification and DNA sequencing success and for species discrimination among the 67 tree and shrub species included in this study. Although we achieved 100% PCR amplification and sequencing success for rbcL and matK, mononucleotide repeats affected sequence quality in matK for some taxonomic groups (e.g. Rosaceae). Species discrimination success ranged from 65% to 71% using tree‐based methods to 86% using BLASTN. The occurrence of known hybrids (diploid and polyploid) and their progenitors on the study site reduced the overall species discrimination success for both loci. This study demonstrates that, even in a floristic context, rbcL and matK alone are insufficient for the discrimination of UK tree species, especially where taxonomically complex groups are present. From a science education perspective, DNA barcoding represents a compelling and accessible platform for the engagement of non‐experts in ongoing research, providing an opportunity for them to contribute authentic scientific data to an international research campaign.     

Estimating correlations among demographic parameters in population models

Estimating correlations among demographic parameters is critical to understanding population dynamics and life‐history evolution, where correlations among parameters can inform our understanding of life‐history trade‐offs, result in effective applied conservation actions, and shed light on evolutionary ecology. The most common approaches rely on the multivariate normal distribution, and its conjugate inverse Wishart prior distribution. However, the inverse Wishart prior for the covariance matrix of multivariate normal distributions has a strong influence on posterior distributions. As an alternative to the inverse Wishart distribution, we individually parameterize the covariance matrix of a multivariate normal distribution to accurately estimate variances (σ²) of, and process correlations (ρ) between, demographic parameters. We evaluate this approach using simulated capture–mark–recapture data. We then use this method to examine process correlations between adult and juvenile survival of black brent geese marked on the Yukon–Kuskokwim River Delta, Alaska (1988–2014). Our parameterization consistently outperformed the conjugate inverse Wishart prior for simulated data, where the means of posterior distributions estimated using an inverse Wishart prior were substantially different from the values used to simulate the data. Brent adult and juvenile annual apparent survival rates were strongly positively correlated (ρ = 0.563, 95% CRI 0.181–0.823), suggesting that habitat conditions have significant effects on both adult and juvenile survival. We provide robust simulation tools, and our methods can readily be expanded for use in other capture–recapture or capture‐recovery frameworks. Further, our work reveals limits on the utility of these approaches when study duration or sample sizes are small.     

DNA barcoding of flowering plants in Sumatra,Indonesia

The rapid conversion of Southeast Asian lowland rainforests into monocultures calls for the development of rapid methods for species identification to support ecological research and sustainable land‐use management. Here, we investigated the utilization of DNA barcodes for identifying flowering plants from Sumatra, Indonesia. A total of 1,207 matK barcodes (441 species) and 2,376 rbcL barcodes (750 species) were successfully generated. The barcode effectiveness is assessed using four approaches: (a) comparison between morphological and molecular identification results, (b) best‐close match analysis with TaxonDNA, (c) barcoding gap analysis, and (d) formation of monophyletic groups. Results show that rbcL has a much higher level of sequence recoverability than matK (95% and 66%). The comparison between morphological and molecular identifications revealed that matK and rbcL worked best assigning a plant specimen to the genus level. Estimates of identification success using best‐close match analysis showed that >70% of the investigated species were correctly identified when using single barcode. The use of two‐loci barcodes was able to increase the identification success up to 80%. The barcoding gap analysis revealed that neither matK nor rbcL succeeded to create a clear gap between the intraspecific and interspecific divergences. However, these two barcodes were able to discriminate at least 70% of the species from each other. Fifteen genera and twenty‐one species were found to be nonmonophyletic with both markers. The two‐loci barcodes were sufficient to reconstruct evolutionary relationships among the plant taxa in the study area that are congruent with the broadly accepted APG III phylogeny.     

Automated analysis of high‐content microscopy data with deep learning

Existing computational pipelines for quantitative analysis of high‐content microscopy data rely on traditional machine learning approaches that fail to accurately classify more than a single dataset without substantial tuning and training, requiring extensive analysis. Here, we demonstrate that the application of deep learning to biological image data can overcome the pitfalls associated with conventional machine learning classifiers. Using a deep convolutional neural network (DeepLoc) to analyze yeast cell images, we show improved performance over traditional approaches in the automated classification of protein subcellular localization. We also demonstrate the ability of DeepLoc to classify highly divergent image sets, including images of pheromone‐arrested cells with abnormal cellular morphology, as well as images generated in different genetic backgrounds and in different laboratories. We offer an open‐source implementation that enables updating DeepLoc on new microscopy datasets. This study highlights deep learning as an important tool for the expedited analysis of high‐content microscopy data.     

Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades‐old dried museum specimens

Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high‐throughput laboratory workflows. The strategy uses hemi‐nested, degenerate, M13‐tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard‐compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, ‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.     

DNA barcoding exposes a case of mistaken identity in the fern horticultural trade

Using cheilanthoid ferns, we provide an example of how DNA barcoding approaches can be useful to the horticultural community for keeping plants in the trade accurately identified. We use plastid rbcL, atpA, and trnG-R sequence data to demonstrate that a fern marketed as Cheilanthes wrightii (endemic to the southwestern USA and northern Mexico) in the horticultural trade is, in fact, Cheilanthes distans (endemic to Australia and adjacent islands). Public and private (accessible with permission) databases contain a wealth of DNA sequence data that are linked to vouchered plant material. These data have uses beyond those for which they were originally generated, and they provide an important resource for fostering collaborations between the academic and horticultural communities. We strongly advocate the barcoding approach as a valuable new technology available to the horticulture industry to help correct plant identification errors in the international trade.     

A reliable DNA barcode reference library for the identification of the North European shelf fish fauna

Valid fish species identification is an essential step both for fundamental science and fisheries management. The traditional identification is mainly based on external morphological diagnostic characters, leading to inconsistent results in many cases. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for a valid identification of 93 North Atlantic fish species originating from the North Sea and adjacent waters, including many commercially exploited species. Neighbour‐joining analysis based on K2P genetic distances formed nonoverlapping clusters for all species with a ≥99% bootstrap support each. Identification was successful for 100% of the species as the minimum genetic distance to the nearest neighbour always exceeded the maximum intraspecific distance. A barcoding gap was apparent for the whole data set. Within‐species distances ranged from 0 to 2.35%, while interspecific distances varied between 3.15 and 28.09%. Distances between congeners were on average 51‐fold higher than those within species. The validation of the sequence library by applying BOLDs barcode index number (BIN) analysis tool and a ranking system demonstrated high taxonomic reliability of the DNA barcodes for 85% of the investigated fish species. Thus, the sequence library presented here can be confidently used as a benchmark for identification of at least two‐thirds of the typical fish species recorded for the North Sea.     

One species in eight: DNA barcodes from type specimens resolve a taxonomic quagmire

Each holotype specimen provides the only objective link to a particular Linnean binomen. Sequence information from them is increasingly valuable due to the growing usage of DNA barcodes in taxonomy. As type specimens are often old, it may only be possible to recover fragmentary sequence information from them. We tested the efficacy of short sequences from type specimens in the resolution of a challenging taxonomic puzzle: the Elachista dispunctella complex which includes 64 described species with minuscule morphological differences. We applied a multistep procedure to resolve the taxonomy of this species complex. First, we sequenced a large number of newly collected specimens and as many holotypes as possible. Second, we used all >400 bp examine species boundaries. We employed three unsupervised methods (BIN, ABGD, GMYC) with specified criteria on how to handle discordant results and examined diagnostic bases from each delineated putative species (operational taxonomic units, OTUs). Third, we evaluated the morphological characters of each OTU. Finally, we associated short barcodes from types with the delineated OTUs. In this step, we employed various supervised methods, including distance‐based, tree‐based and character‐based. We recovered 658 bp barcode sequences from 194 of 215 fresh specimens and recovered an average of 141 bp from 33 of 42 holotypes. We observed strong congruence among all methods and good correspondence with morphology. We demonstrate potential pitfalls with tree‐, distance‐ and character‐based approaches when associating sequences of varied length. Our results suggest that sequences as short as 56 bp can often provide valuable taxonomic information. The results support significant taxonomic oversplitting of species in the Elachista dispunctella complex.     

A DNA mini‐barcode for land plants

Small portions of the barcode region – mini‐barcodes – may be used in place of full‐length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini‐barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini‐barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30 472)]. PCR amplification for all mini‐barcodes, as estimated by validated electronic simulation, was successful for 90.2–99.8% of species. Overall Sanger sequence quality for mini‐barcodes was very low – the best mini‐barcode tested produced sequences of adequate quality (B₂₀ ≥ 0.5) for 74.5% of samples. The majority of mini‐barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini‐barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini‐barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini‐barcode D (F52/R193).     

An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.