Barcode Sequences Research Articles

The tetracycline resistome is shaped by selection for specific resistance mechanisms by each antibiotic generation

The history of clinical resistance to tetracycline antibiotics is characterized by cycles whereby the deployment of a new generation of drug molecules is quickly followed by the discovery of a new mechanism of resistance. This suggests mechanism-specific selection by each tetracycline generation; however, the evolutionary dynamics of this remain unclear. Here, we evaluate 24 recombinant Escherichia coli strains expressing tetracycline resistance genes from each mechanism (efflux pumps, ribosomal protection proteins, and enzymatic inactivation) in the context of each tetracycline generation. We employ a high-throughput barcode sequencing protocol that can discriminate between strains in mixed culture and quantify their relative abundances. We find that each mechanism is preferentially selected for by specific antibiotic generations, leading to their expansion. Remarkably, the minimum inhibitory concentration associated with individual genes is secondary to resistance mechanism for inter-mechanism relative fitness, but it does explain intra-mechanism relative fitness. These patterns match the history of clinical deployment of tetracycline drugs and resistance discovery in pathogens.

Delimiting Species-Prospects and Challenges for DNA Barcoding.

Discovering, describing and cataloguing global species diversity remains a fundamental challenge both for biodiversity research and for the management and conservation of biodiversity. Among animals, the challenge is particularly acute within the arthropods, which comprise approximately 85% of all described animals, with approximately 1 million described species. The true number of arthropod species is estimated to be in excess of 10 million species. This estimate is likely to be revised upward in the light of global DNA barcode sequencing initiatives that are cataloguing unprecedented levels of cryptic or overlooked diversity. The scale of diversity that is being recovered with barcode sequencing places further strain on a taxonomic system confronted by ever-limited global taxonomic capacity to verify and describe new species. It is predicted that the number of novel operational taxonomic units delimited by barcode sequencing is likely to eclipse the number of species described by Linnean taxonomy by as early as 2029. Unless addressed, this may see an increasing proportion of arthropod species falling outside of protective legislative frameworks as a consequence of their lack of formal description. Confronted with this challenge, there is increasing, but controversial, acceptance of species delimitation and species description based on barcode sequence clustering thresholds. In response to the evolving controversy surrounding this issue, it is both timely and important to identify and clarify prospects and challenges for DNA barcoding, with a specific focus on species delimitation to address important shortfalls and impediments in biodiversity research.

Deterministic genetic barcoding for multiplexed behavioral and single-cell transcriptomic studies

Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression and cellular heterogeneity within tissues and have enabled the construction of transcriptomic cell atlases. However, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM enables positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that TaG-EM barcodes can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to multiplex and reliably annotate single-cell transcriptomic experiments.

Deterministic genetic barcoding for multiplexed behavioral and single-cell transcriptomic studies.

Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression and cellular heterogeneity within tissues and have enabled the construction of transcriptomic cell atlases. However, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM enables positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that TaG-EM barcodes can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to multiplex and reliably annotate single-cell transcriptomic experiments.

The best of two worlds: toward large‐scale monitoring of biodiversity combining COI metabarcoding and optimized parataxonomic validation

In a context of unprecedented insect decline, it is critical to have reliable monitoring tools to measure species diversity and their dynamic at large‐scales. High‐throughput DNA‐based identification methods, and particularly metabarcoding, were proposed as an effective way to reach this aim. However, these identification methods are subject to multiple technical limitations, resulting in unavoidable false‐positive and false‐negative species detection. Moreover, metabarcoding does not allow a reliable estimation of species abundance in a given sample, which is key to document and detect population declines or range shifts at large scales. To overcome these obstacles, we propose here a human‐assisted molecular identification (HAMI) approach, a framework based on a combination of metabarcoding and image‐based parataxonomic validation of outputs and recording of abundance. We assessed the advantages of using HAMI over the exclusive use of a metabarcoding approach by examining 492 mixed beetle samples from a biodiversity monitoring initiative conducted throughout France. On average, 23% of the species are missed when relying exclusively on metabarcoding, this percent being consistently higher in species‐rich samples. Importantly, on average, 20% of the species identified by molecular‐only approaches correspond to false positives linked to cross‐sample contaminations or mis‐identified barcode sequences in databases. The combination of molecular methodologies and parataxonomic validation in HAMI significantly reduces the intrinsic biases of metabarcoding and recovers reliable abundance data. This approach also enables users to engage in a virtuous circle of database improvement through the identification of specimens associated with missing or incorrectly assigned barcodes. As such, HAMI fills an important gap in the toolbox available for fast and reliable biodiversity monitoring at large scales.

High throughput single cell metagenomic sequencing with semi-permeable capsules: unraveling microbial diversity at the single-cell level in sewage and fecal microbiomes

Single-cell sequencing may serve as a powerful complementary technique to shotgun metagenomics to study microbiomes. This emerging technology allows the separation of complex microbial communities into individual bacterial cells, enabling high-throughput sequencing of genetic material from thousands of singular bacterial cells in parallel. Here, we validated the use of microfluidics and semi-permeable capsules (SPCs) technology (Atrandi) to isolate individual bacterial cells from sewage and pig fecal samples. Our method involves extracting and amplifying single bacterial DNA within individual SPCs, followed by combinatorial split-and-pool single-amplified genome (SAG) barcoding and short-read sequencing. We tested two different sequencing approaches with different numbers of SPCs from the same sample for each sequencing run. Using a deep sequencing approach, we detected 1,796 and 1,220 SAGs, of which 576 and 599 were used for further analysis from one sewage and one fecal sample, respectively. In shallow sequencing data, we aimed for 10-times more cells and detected 12,731 and 17,909 SAGs, of which we used 2,456 and 1,599 for further analysis for sewage and fecal samples, respectively. Additionally, we identified the top 10 antimicrobial resistance genes (ARGs) in both sewage and feces samples and linked them to their individual host bacterial species.

A genome-wide atlas of human cell morphology.

A key challenge of the modern genomics era is developing empirical data-driven representations of gene function. Here we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-wide genotype-phenotype maps comprising CRISPR-Cas9-based knockouts of >20,000 genes in >30 million cells. Our optical pooled cell profiling platform (PERISCOPE) combines a destainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This perturbation atlas comprises high-dimensional phenotypic profiles of individual cells with sufficient resolution to cluster thousands of human genes, reconstruct known pathways and protein-protein interaction networks, interrogate subcellular processes and identify culture media-specific responses. Using this atlas, we identify the poorly characterized disease-associated TMEM251/LYSET as a Golgi-resident transmembrane protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes. In sum, this perturbation atlas and screening platform represents a rich and accessible resource for connecting genes to cellular functions at scale.

Typification of the Economically Important Species Thyreophagus entomophagus (Acari: Astigmata: Acaridae) Used for the Industrial Production of Predatory Mites: The Designation of a Neotype with Detailed Morphological and DNA Sequence Data

The mite Thyreophagus entomophagus is a cosmopolitan species of significant economic importance in biocontrol applications, serving as a factitious prey for the mass rearing of predatory mites. This species has been reported from a variety of habitats. However, the taxonomic reliability of its name is questionable due to inconsistencies in historical species identifications, the absence of type specimens, and misidentified GenBank sequences. Here, to address these issues and to standardize the nomenclature, we redescribe Thyreophagus entomophagus based on a commercial culture with known COX1 barcoding sequence data and designate a neotype from this culture. As part of delimiting the species boundaries of Th. entomophagus, the question of whether this species forms heteromorphic deutonymphs is particularly important. While the literature suggests that most populations lack them, at least one population in Germany has been reported to produce heteromorphic deutonymphs. However, after careful examination, we identified this population as a new species, Thyreophagus holda, indicating that previous identifications of this population as Th. entomophagus were incorrect. The absence of the heteromorphic deutonymphal stage is a beneficial trait for mass production, as it simplifies the life cycle by eliminating the energetically costly heteromorphic deutonymph. Our preliminary molecular phylogenetic analyses of Th. entomophagus and other species of Thyreophagus indicate that the loss of heteromorphic deutonymphs and the emergence of asexual reproduction (another beneficial trait for mass production) are derived traits that arose after the divergence of the most recent common ancestor of Thyreophagus. These insights enhance our understanding of the evolutionary traits that increase the effectiveness of Th. entomophagus and related species in biocontrol settings. Our study points to the need for additional bioprospecting efforts to identify new candidate species for biocontrol that possess both asexual reproduction and the absence of heteromorphic deutonymphs.

Massively parallel barcode sequencing revealed the interchangeability of capsule transporters in Streptococcus pneumoniae.

Multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) family transporters are essential in glycan synthesis, flipping lipid-linked precursors across cell membranes. Yet, how they select their substrates remains enigmatic. Here, we investigate the substrate specificity of the MOP transporters in the capsular polysaccharide (CPS) synthesis pathway in Streptococcus pneumoniae. These capsule flippases collectively transport more than 100 types of capsule precursors. To determine whether they can substitute for one another, we developed a high-throughput approach to systematically examine nearly 6000 combinations of flippases and substrates. CPS flippases fall into three groups: relaxed, type-specific, and strictly specific. Cargo size and CPS acetylation affect transport, and we isolated additional gain-of-function flippase variants that can substitute for the peptidoglycan flippase YtgP (MurJ). We also showed that combining flippase variants in a single cassette allows various CPS precursors to be flipped, which may aid glycoengineering. This study reveals that MOP flippases exhibit broad specificity, shaping the evolution of glycan synthesis.

Diversity and seasonality of ectoparasite burden on two species of Madagascar fruit bat, Eidolon dupreanum and Rousettus madagascariensis.

Bats are important reservoir hosts for a variety of microparasites, some of which are transmitted by ectoparasite vectors that include mites, fleas, lice, ticks, and bat flies (families Nycteribiidae and Streblidae). All of these ectoparasite taxa are known to parasitize two endemic fruit bats of Madagascar, Eidolon dupreanum and Rousettus madagascariensis. We aimed to describe the diversity of ectoparasite infestation for both bat species through morphological observation and DNA barcoding and elucidate ecological and climatic correlates of seasonal nycteribiid parasitism of these hosts. Live E. dupreanum and R. madagascariensis fruit bats were captured monthly in northern and central-eastern Madagascar from 2013-2020. Ectoparasites on all captured bats were counted and identified in the field, then collected into ethanol. Field identification of a subset of samples were confirmed via microscopy and DNA barcoding of the cytochrome C oxidase subunit 1 (COI) and 18S genes. The seasonal abundance of nycteribiid bat flies on both host bats was analyzed using generalized additive models, and the role of climate in driving this seasonality was assessed via cross-correlation analysis combined with generalized linear models. Phylogenetic trees were generated to compare COIand 18S sequences of Madagascar nycteribiid and streblid bat flies with available reference sequences from GenBank. Ectoparasites corresponding to four broad taxa (mites, ticks, fleas, and bat flies) were recovered from 628 of 873 E. dupreanum and 831 of 862 R. madagascariensis . E. dupreanum were most commonly parasitized by Cyclopodia dubia nycteribiids and R. madagascariensis by Eucampsipoda madagascariensis nycteribiids or Megastrebla wenzeli streblids. We observed significant seasonality in nycteribiid abundance on both bat hosts, which varied by bat sex and was positively correlated with lagged temperature, precipitation, and humidity variables. Barcoding sequences recovered for all three bat fly species grouped with previously reported sequences, confirming morphological species identification. Our study contributes the first DNA barcodes of any kind reported for M. wenzeli and the first 18S barcodes for C. dubia . This study explores the diversity and abundance of ectoparasite burdens in two Malagasy fruit bat species, highlighting the importance of seasonal ecology and the influence of climate variables on parasitism, which correlates with resource availability.

A comparison and evaluation of strategies for bulk sequencing of mitogenomes from museum collections

Abstract Collecting mitochondrial genome sequences from museum collections has garnered increasing attention in recent years. Currently, several sequencing strategies have been proposed for this purpose, including direct DNA pooling, DNA labelling and mtDNA enrichment. It is important to understand the advantages and limitations of these methods before embarking on large‐scale sequencing efforts. However, there is currently no comprehensive comparison and evaluation of the performance of these sequencing methods. In this study, we evaluated the performance of four different strategies for sequencing mitogenomes by comparing reagent expense, experiment time, bioinformatics time, and the length and the base error rate of the obtained mitochondrial sequences. The four sequencing strategies mainly differ in: (1) whether to label the DNA of each specimen and (2) whether to perform mtDNA enrichment. To reduce experiment difficulty and expenses, we presented an ‘in‐situ’ DNA labelling protocol for cost‐effectively ligating barcode sequences to DNA samples and used PCR‐generated baits for mtDNA enrichment. The widely adopted ‘direct pooling’ sequencing strategy is the fastest but performs the worst among the four strategies. Of the 96 samples analysed, this approach yields averagely only 670 bp of mitochondrial sequences per sample. The most complicated sequencing strategy, which labels the DNA of each specimen and performs mtDNA enrichment, performs the best, producing mitogenome sequences of ~9.3 kb per sample. The sequencing accuracy is highly related to the sequencing depths of mtDNA but not the storage time of samples. We hope that our comparisons will assist researchers in selecting suitable strategies for large‐scale sequencing of mitogenomes from museum specimens, thereby advancing museomics.

A new species of the genus Oiketicoides Heylaerts, 1885 (Lepidoptera, Psychidae) from Korea with its natural parasitoid enemy.

Oiketicoidesgohadoensis Roh & Lee, sp. nov. is described as new to science. The morphology of male adult, including genitalia, is described, and DNA barcodes for precise identification of the species are provided. A parasitoid, Neophryxepsychidis Townsend, 1916 (Diptera, Tachinidae) of O.gohadoensis is also reported for the first time in Korea, together with its DNA barcode sequence.

Development of a new primer cocktail for improved DNA barcoding of brachyuran crabs (Decapoda: Brachyura)

Abstract DNA barcoding has revolutionized species identification and molecular phylogeny, offering immense potential for elucidating the diversity of brachyuran crabs. We explored the utility of a new primer cocktail-based approach for amplifying DNA barcode regions of mitochondrial cytochrome c oxidase I (CO1) from 28 specimens representing brachyuran species across 21 major families collected along the Korean coast. The primer cocktail, comprising three forward and two reverse degenerate primers with M13 adapter tails, exhibited superior PCR amplification efficiency, being 100% successful. Sequencing of the PCR products yielded high-quality, 726 bp, DNA barcode sequences, facilitating accurate species identification with 23 out of 28 specimens matching precisely with their morphological taxonomy. Barcoding gap analysis revealed a clear gap between mean intra- and interspecific K2P distances, with a 148-fold difference, confirming the reliability of CO1 barcodes for achieving 100% success in species identification within the dataset. The study highlights the effectiveness and reliability of the new primer cocktail as a robust molecular marker for DNA barcoding of brachyuran crabs, essential for ecological investigations and conservation endeavors, and elucidating the taxonomic complexities among them.

Building a reliable 16S mini-barcode library of wild bees from Occitania, south-west of France.

DNA barcoding and metabarcoding are now powerful tools for studying biodiversity and especially the accurate identification of large sample collections belonging to diverse taxonomic groups. Their success depends largely on the taxonomic resolution of the DNA sequences used as barcodes and on the reliability of the reference databases. For wild bees, the barcode sequences coverage is consistently growing in volume, but some incorrect species annotations need to be cared for. The COI (Cytochrome Oxydase subunit 1) gene, the most used in barcoding/metabarcoding of arthropods, suffers from primer bias and difficulties for covering all wild bee species using the classical Folmer primers. We present here a curated database for a 250 bp mini-barcode region of the 16S rRNA gene, suitable for low-cost metabarcoding wild bees in applications, such as eDNA analysis or for sequencing ancient or degraded DNA. Sequenced specimens were captured in Occitania (south-west of France) and morphologically identified by entomologists, with a total of 530 individuals belonging to 171 species and 19 genera. A customised workflow including distance-tree inferences and a second round of entomologist observations, when necessary, was used for the validation of 348 mini-barcodes covering 148 species. Amongst them, 93 species did not have any 16S reference barcode available before our contribution. This high-quality reference library data are freely available to the scientific community, with the aim of facilitating future large-scale characterisation of wild bee communities in a context of pollinators' decline.

Identification of Starmerella aleppica f.a., sp. nov. and large indels in the rRNA cistron that split the Starmerella genus.

Yeast strains representing a novel asexual ascomycetous species were isolated from seven Malva sylvestris flowers. Sequencing of the chromosomal regions coding for the D1/D2 domains of the large subunit ribosomal RNA, the ITS1-5.8S-ITS2 segments and parts of the gene coding for the small subunit ribosomal RNA showed that the isolates were conspecific. Comparative analysis of these sequences and the corresponding sequences of the type strains of ascomycetous yeasts revealed that the strains represent a hitherto undescribed species belonging to the sensu stricto subclade of the genus Starmerella. The new species is osmotolerant and can develop invasive pseudohyphae, but does not form spores. For the new species, the name Starmerella aleppica f.a. (forma asexualis) is proposed. The holotype, preserved in a metabolically inactive state, is CBS 12960T (extype cultures: 2-1361 and CCY 90-2-1, NCAIM Y.02123). The GenBank accession numbers of barcode sequences are JX515983 (D1/D2 domain), JX515985 (ITS1-5.8S-ITS2 and partial 18S rRNA gene), PQ613837 (TEF1 partial sequence) and PQ613838 (RPB2 partial sequence). MycoBank: MB855459. The analysis of the D1/D2 and internal transcribed spacer (ITS) sequences of the type strains of species of the genus identified multiple multinucleotide indels that can be used as taxonomic markers (InDel markers). The indel patterns of the subclades are very different and homogeneous within the subclades. This result reinforces the idea raised, but also refuted, in previous studies that the Starmerella subclades may represent different genera.

Testing Phylogenetic Placement Accuracy of DNA Barcode Sequences on a Fish Backbone Tree: Implications of Backbone Tree Completeness and Species Representation.

Advancements in DNA sequencing technology have facilitated the generation of a vast number of DNA sequences, posing opportunities and challenges for constructing large phylogenetic trees. DNA barcode sequences, particularly COI, represent extensive orthologous sequences suitable for phylogenetic analysis. Phylogenetic placement analysis offers a promising method to integrate COI data into tree-building efforts, yet the impacts of backbone tree completeness and species composition remain under-explored. Using a dataset comprising 27 genes and 4520 species of bony fishes, we assessed the accuracy of phylogenetic inference by "placing" COI sequences onto backbone trees. The backbone tree completeness was varied by subsampling 20%, 40%, 60%, 80%, and 99% of the total species separately, followed by placement of those missing species based on their COI sequences using software packages EPA-ng and APPLES. We also compared the effects of biased, random, and stratified sampling strategies; the latter ensured the representation of all major lineages (Family) of bony fish. Our findings indicate that the placement accuracy is consistently high across all levels of backbone tree completeness, where 70%-78% missing species are correctly placed (by EPA-ng) in the same locations as the reference tree derived from the complete data. High completeness produces slightly high placement accuracy, although in many cases the differences are nonsignificant. For example, at the 99% completeness level with stratified sampling, EPA-ng placed 78% missing species correctly, and when only considering placement with high confidence (LWR > 0.9), the percentage is 87%. Additionally, stratified sampling outperforms random sampling in most cases, and biased sampling has the worst performance. The likelihood-based EPA-ng consistently provide higher accurate placements than the distance-based APPLES. In conclusion, COI-based placement analysis represents a potential route of using the available vast barcoding data for building large phylogenetic trees.

In Vitro conservation and genetic diversity analysis of rare species Ribes janczewskii

Ribes janczewskii is a rare and valuable plant known for its resistance to spring frosts, pests, and diseases. It is used in hybridization to develop resistant currant varieties but is on the verge of extinction, listed in Kazakhstan Red Book. This study developed a micropropagation and slow-growth storage protocol for conservation. Genotypes were identified through DNA barcode analysis (rbcL, ITS, and matK) and sequences uploaded to the National Center for Biotechnology Information database. Genetic diversity was assessed using iPBS primers, generating 98 fragments with 88–94% polymorphic bands. Biochemical analysis of fruits showed vitamin C content from 4.64 to 5.61 mg/100 g, vitamin E from 2.26 to 3.16 mg/100 g, vitamin B5 from 3.18 to 4.93 mg/100 g, and quercetin up to 12.5 mg/100 g. Micropropagation stages were optimized with 12% hydrogen peroxide for surface sterilization, achieving up to 73.3% explant viability. Effective hormonal combinations for in vitro culture included WPM with BAP 0.2 mg L−1 and GA 0.5 mg L−1, and for propagation, BAP 0.25 mg L−1, GA 0.5 mg L−1, and IBA 0.5 mg L−1. Mannitol (20 g L−1) was used for slow-growth storage, keeping explants viable for 4 months without re-cultivation.

From Species to Varieties: How Modern Sequencing Technologies Are Shaping Medicinal Plant Identification.

Modern sequencing technologies have transformed the identification of medicinal plant species and varieties, overcoming the limitations of traditional morphological and chemical approaches. This review explores the key DNA-based techniques, including molecular markers, DNA barcoding, and high-throughput sequencing, and their contributions to enhancing the accuracy and reliability of plant identification. Additionally, the integration of multi-omics approaches is examined to provide a comprehensive understanding of medicinal plant identity. The literature search for this review was conducted across databases such as Google Scholar, Web of Science, and PubMed, using keywords related to plant taxonomy, genomics, and biotechnology. Inclusion criteria focused on peer-reviewed studies closely related to plant identification methods and techniques that contribute significantly to the field. The review highlights that while sequencing technologies offer substantial improvements, challenges such as high costs, technical expertise, and the lack of standardized protocols remain barriers to widespread adoption. Potential solutions, including AI-driven data analysis and portable sequencers, are discussed. This review provides a comprehensive overview of molecular techniques, their transformative impact, and future perspectives for more accurate and efficient medicinal plant identification.

New ecological insights on wild pollinator Andrena hesperia

A nesting aggregation of Andrena hesperia in Bologna (Italy) was studied to characterize this bee’s ecology, phenology and interactions with the environment. Andrena hesperia adults emerged between the end of March and the middle of April, displaying univoltine, protandrous phenology. The average resistance to heat stupor of A. hesperia females was 42.53 ± 13.77 minutes at 40°C. Parasites Nomada facilis and Bombylius canescens were associated with the nests. DNA barcode sequences (COI gene) of A. hesperia and N. facilis were sequenced and deposited in GenBank. The gut microbiota of newly emerged individuals was dominated by Bacillota (Lactobacillus and Fructobacillus) and Pseudomonadota (Snodgrassella alvei and Gilliamella). The pollen carried by A. hesperia females was identified morphologically as belonging for the most part to the Asteraceae family. The pathogens detected on A. hesperia showed different infection loads in newly-emerged individuals and foraging adults. This is the first time that comprehensive information on A. hesperia is reported, and it will hopefully foster further studies on this wild bee.

Automated Design of Oligopools and Rapid Analysis of Massively Parallel Barcoded Measurements.

Oligopool synthesis and next-generation sequencing enable the construction and characterization of large libraries of designed genetic parts and systems. As library sizes grow, it becomes computationally challenging to optimally design large numbers of primer binding sites, barcode sequences, and overlap regions to obtain efficient assemblies and precise measurements. We present the Oligopool Calculator, an end-to-end suite of algorithms and data structures that rapidly designs many thousands of oligonucleotides within an oligopool and rapidly analyzes many billions of barcoded sequencing reads. We introduce several novel concepts that greatly increase the design and analysis throughput, including orthogonally symmetric barcode design, adaptive decision trees for primer design, a Scry barcode classifier, and efficient read packing. We demonstrate the Oligopool Calculator's capabilities across computational benchmarks and real-data projects, including the design of over four million highly unique and compact barcodes in 1.2 h, the design of universal primer binding sites for one million 200-mer oligos in 15 min, and the analysis of about 500 million deep sequencing reads per hour, all on an 8-core desktop computer. Overall, the Oligopool Calculator accelerates the creative use of massively parallel experiments by eliminating the computational complexity of their design and analysis.