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Accurate taxonomic species identification is essential to the study of mammals. Despite this necessity, rapid and accurate identification of cryptic, understudied, and elusive mammals remains challenging. Traditional barcoding of mitochondrial genes is standard for molecular identification but requires time-consuming wet-lab methodologies. Recent bioinformatic advancements for nanopore sequencing data offer exciting opportunities for noninvasive and field-based identification of mammals. Nanopore adaptive sampling (NAS), a polymerase chain reaction (PCR)-free method, selectively sequences regions of DNA according to user-specified reference databases. Here, we utilized NAS to enrich mammalian mitochondrial genome sequencing to identify species. Fecal DNA extractions were sequenced from 9 mammals, several collected in collaboration with Minnesota Tribal Nations, to demonstrate utility for NAS barcoding of noninvasive samples. By mapping to the entire National Center for Biotechnology Information mammalian mitochondrial reference genome database and bioinformatically analyzing highly similar matches, we successfully produced species identifications for all fecal samples. Eight of 9 species identifications matched previous PCR or animal/fecal appearance-based identifications. For the ninth species, our genetic data indicate a misidentification stemming from the original study. Our approach has a range of applications—particularly in field-based wildlife research, conservation, disease surveillance, and monitoring of wildlife trade. Of importance to Minnesota tribes is invasive species monitoring, detections, and confirmation as climate impacts cause changes in biodiversity and shifts in species distributions. The rapid assessment techniques described here will be useful as new introductions and range expansions of native and invasive species may first be detected by the presence of signs such as scat rather than direct observations and will be helpful for chronically understaffed tribal natural resources agencies. [ABSTRACT FROM AUTHOR]

The Acorus calamus group, or sweet flag, includes important medicinal plants and is classified into three species: A. americanus (diploid), A. verus (tetraploid), and A. calamus (sterile triploid of hybrid origin). Members of the group are famous as components of traditional Indian medicine, and early researchers suggested the origin of the sweet flag in tropical Asia. Subsequent research led to an idea of the origin of the triploid A. calamus in the Amur River basin in temperate Asia, because this was the only region where both diploids and tetraploids were known to co-occur and be capable of sexual reproduction. Contrary to this hypothesis, triploids are currently very rare in the Amur basin. Here, we provide the first evidence that all three species occur in Kazakhstan. The new records extend earlier data on the range of A. verus for c. 1800 km. Along the valley of the Irtysh River in Kazakhstan and the adjacent Omsk Oblast of Russia, A. verus is recorded in the south, A. americanus in the north, and A. calamus is common in between. We propose the Irtysh River valley as another candidate for a cradle of the triploid species A. calamus. It is possible that the range of at least one parent species (A. americanus) has contracted through competition with its triploid derivative species, for which the Irtysh River floods provide a tool for downstream range expansion. We refine our earlier data and show that the two parent species have non-overlapping ranges of variation in a quantitative metric of leaf aerenchyma structure. [ABSTRACT FROM AUTHOR]

Four species of the genus Sticta are described as new from Bolivia, based on morphological examination and phylogenetic analysis of the fungal ITS barcoding marker. Additionally, two species are reported as new to Bolivia (their identification confirmed by molecular data) and one previously reported species is confirmed by molecular data for the first time. Detailed morphological and anatomical descriptions are provided for all new species. Two of the new species, S. isidiolobulata Ossowska, B. Moncada, Lücking & Kukwa and S. madidiensis Ossowska, B. Moncada, Lücking & Kukwa belong to clade I, as defined in previous studies. In contrast, S. montepunkuensis Ossowska, B. Moncada, Lücking & Kukwa and S. macrolobata Ossowska, B. Moncada, Lücking & Kukwa, also described here as new to science, belong to clade III. Sticta isidiolobulata has an irregular to suborbicular thallus of medium size, with isidia developing into spathulate lobules, cyanobacterial photobiont and apothecia with entire to weakly-crenate margins. The large irregular thallus of the cyanobacteria-associated S. macrolobata has broad lobes, apothecia with verrucous to tomentose margins and cyphellae with raised margins, whereas S. madidiensis has a medium-sized, palmate to irregular thallus with a stipe, but without vegetative propagules and apothecia. Sticta montepunkuensis has large and irregular thalli with green algae as photobiont, apothecia with crenate to verrucous margins and urceolate cyphellae with a wide pore and a scabrid basal membrane. Two species, S. beauvoisii Delise and S. riparia Merc.-Díaz are reported as new to Bolivia (the latter also as new to South America) and belong to clade III. Sticta tomentosa (Sw.) Ach., species confirmed from Bolivia by molecular data, belongs to clade II. Sticta beauvoisii is characterised by a smooth yellowish-brown upper surface with darker apices and abundant, marginal isidia and a brown lower surface with golden-chocolate brown primary tomentum and sparse, golden-brown rhizines. Sticta riparia has a strongly branched thallus, with undulate lobes and abundant, marginal, palmate, grey to dark brown phyllidia and greyish-brown lower surface with the primary tomentum absent towards the margins. Sticta tomentosa has palmate, bluish thalli with white cilia and abundant, submarginal apothecia and creamy-white lower surface with a sparse, white primary tomentum. [ABSTRACT FROM AUTHOR]

Simple Summary: The chigger mite Neoschoengastia gallinarum (Hatori, 1920) is a parasite that feeds on the skin tissue of birds across multiple countries in Southeast and East Asia. In domestic chickens, heavy infestations with this mite can lead to skin irritation and damage to the carcass, reducing economic value. In this study, we collected N. gallinarum samples from wild birds of conservation concern and domestic chickens in Peninsular Malaysia and Thailand. Sequence analyses of three genes from the mites were compared across four Malaysian populations, one Thai population, and previously published sequences from southeastern China. A variety of methods were applied to classify these sequences and determine the extent of interbreeding between populations. These methods agreed in identifying three clusters of sequences by country of origin, although there was partial overlap between Thailand and China. The populations from Malaysia and Thailand appear to be reproductively isolated from one another and may represent distinct species with almost identical morphological features, except for leg length. Further studies are required to determine if these genetic dissimilarities are accompanied by distinct ecological, behavioural, or pathological differences in N. gallinarum in different regions of Asia. Neoschoengastia gallinarum is widely distributed in Asia, preferentially parasitising birds, and heavy infestations have clinical impacts on domestic fowl. In common with other trombiculid mites, the genetic diversity and potential variation in host preferences or pathology induced by N. gallinarum are poorly understood. This study aimed to unravel the geographical variation and population structure of N. gallinarum collected from galliform birds in Peninsular Malaysia and Thailand by inference from concatenated mitochondrial-encoded cytochrome c oxidase subunit I (COI), and nuclear-encoded internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA gene sequences, including a comparison with previously published data from southeastern China. Our multi-locus sequence analysis revealed three monophyletic clades comprising (A) specimens from Peninsular Malaysia, (B) the samples from Thailand together with a minority of Chinese sequences, and (C) the majority of sequences from China. Similarly, most species delimitation approaches divided the specimens into three operational taxonomic units. Analysis of molecular variance revealed 96.41% genetic divergence between Malaysian and Thai populations, further supported by the absence of gene flow (Nm = 0.01). In conclusion, despite the two countries sharing a land border, populations of N. gallinarum from Peninsular Malaysia and Thailand appear to be genetically segregated and may represent distinct cryptic species. [ABSTRACT FROM AUTHOR]

The first records of Parmelia ernstiae from Madeira, as well as the new localities of P. encryptata from Poland and P. sulcata from Chile, are presented. All records are confirmed by molecular data, BLAST search, and haplotype network analysis of the nucITS rDNA sequences. The discussion on the morphology and secondary chemistry of all three species is provided with notes on their distribution and similar taxa. Parmelia encryptata is a rarely reported cryptic species that is morphologically indistinguishable from P. sulcata , and its identification requires molecular data. New localities of P. encryptata from northern Poland suggest that the taxon may be widespread in the country and not confined to large forests but also present in open areas on free-standing trees. Moreover, a new nucITS rDNA haplotype of the species is reported. Parmelia ernstiae is a near-cryptic species, morphologically and chemically very similar to P. saxatilis and P. serrana . Two specimens of the species from Madeira have sublinear lobes with marginal and laminal isidia, unlike most specimens of P. ernstiae, having short, broad lobes with laminal isidia, and thus are morphologically closer to P. saxatilis in lobe shape and to P. serrana in placement of isidia. However, the BLAST search and haplotype analysis of nucITS rDNA sequences placed these specimens in P. ernstiae . Parmelia sulcata , a common species in the Northern Hemisphere, is rare elsewhere, and only two records confirmed by molecular data were known from South America. The nucITS rDNA haplotype of two new and two previous records of the species represent the most common haplotype of the species known from the Northern Hemisphere.

Nanipora Miyazaki & Reimer, 2015 is a recently described monotypic octocoral genus belonging to the family Helioporidae (class Octocorallia). Nanipora kamurai Miyazaki & Reimer, 2015 was formally described from shallow coral reefs around Zamami Island, within Kerama-shoto National Park, in Okinawa Prefecture, southern Japan. To date, Nanipora has been reported from two other locations in Japan: N. aff. kamurai from a shallow inner lagoon at Iriomotejima Island, and Nanipora cf. kamurai from the coral reef surrounding a shallow CO2 vent at Iotorishima Island, both in Okinawa Prefecture. Additionally, Nanipora cf. kamurai has been reported from three locations outside Japan: in shallow seagrass meadows at Dongsha Atoll in the South China Sea, on coral reefs at Koh Tao in Thailand and from a reef in Toboso, central Philippines. Finally, N. kamurai has been detected in environmental DNA samples from the Rowley Shoals off northwestern Australia. As a ‘living fossil’ and also apparently common in the low pH environment of Iotorishima I., a clearer understanding of the distribution of the genus should aid in better understanding of its ecology. Here, we report three new additional records from the Ryukyu Archipelago where Nanipora colonies were found, resulting in ten total records of Nanipora. Molecular phylogenetic analyses utilizing cytochrome oxidase subunit I (COI), mitochondrial mismatch repair protein (mtMutS), and the internal transcribed spacer region (ITS) sequences of specimens from two of these new locations combined with additional sequences from previously collected specimens from Dongsha, Iotorishima, and Iriomotejima showed evidence of three closely related groups within Nanipora, warranting future investigations into the species diversity of this genus. [ABSTRACT FROM AUTHOR]

Abstract Background Blood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources. Methods We used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method. Results Complete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively. Conclusions Our findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel ‘phylogenetic capture’ methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens. Graphical Abstract

Neoschoengastia gallinarum is widely distributed in Asia, preferentially parasitising birds, and heavy infestations have clinical impacts on domestic fowl. In common with other trombiculid mites, the genetic diversity and potential variation in host preferences or pathology induced by N. gallinarum are poorly understood. This study aimed to unravel the geographical variation and population structure of N. gallinarum collected from galliform birds in Peninsular Malaysia and Thailand by inference from concatenated mitochondrial-encoded cytochrome c oxidase subunit I (COI), and nuclear-encoded internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA gene sequences, including a comparison with previously published data from southeastern China. Our multi-locus sequence analysis revealed three monophyletic clades comprising (A) specimens from Peninsular Malaysia, (B) the samples from Thailand together with a minority of Chinese sequences, and (C) the majority of sequences from China. Similarly, most species delimitation approaches divided the specimens into three operational taxonomic units. Analysis of molecular variance revealed 96.41% genetic divergence between Malaysian and Thai populations, further supported by the absence of gene flow (Nm = 0.01). In conclusion, despite the two countries sharing a land border, populations of N. gallinarum from Peninsular Malaysia and Thailand appear to be genetically segregated and may represent distinct cryptic species.

Bryophilous myxomycetes are found mainly in humid mountainous regions, particularly in the Ukrainian Carpathians. Over the last three seasons, three species from this group (Colloderma oculatum, Diderma tigrinum, and Lamproderma sp.) have been discovered in Slobozhanskyi National Nature Park, a lowland region in the forest-steppe zone of north-eastern Ukraine. These finds indicate an expansion of the distribution range of bryophilous myxomycetes beyond their typical montane and boreal habitats. The unidentified species of the genus Lamproderma, related to L. muscicola, exhibits distinct morphological and molecular characteristics sufficient to consider it a separate species. [ABSTRACT FROM AUTHOR]

Based on a study of 255 collections from four continents and four floristic kingdoms, we describe 15 new species of the genus Lycogala. The new species, all morphologically close to L. epidendrum, L. exiguum, and L. confusum, differ from each other by the structure of the peridium and, in some cases, also by the color of the fresh spore mass and the ornamentation of the capillitium and spores. Species delimitation is confirmed by two independently inherited molecular markers, as well as previously performed tests of reproductive isolation and genetic distances. We studied authentic material of L. exiguum and L. confusum and found fresh specimens of these species, which allowed us to obtain molecular barcodes and substantiate the separation of new species from these taxa. We propose to retain the name L. epidendrum for the globally most abundant species, for which we provide a more precise description and a neotypification. Two formerly described species, L. leiosporum and L. fuscoviolaceum, we consider to be dubious. We do not recognize the species L. terrestre. [ABSTRACT FROM AUTHOR]

The recent increase in international fish trade leads to the need for improving the traceability of fishery products. In relation to this, consistent monitoring of the production chain focusing on technological developments, handling, processing and distribution via global networks is necessary. Molecular barcoding has therefore been suggested as the gold standard in seafood species traceability and labelling. This review describes the DNA barcoding methodology for preventing food fraud and adulteration in fish. In particular, attention has been focused on the application of molecular techniques to determine the identity and authenticity of fish products, to discriminate the presence of different species in processed seafood and to characterize raw materials undergoing food industry processes. In this regard, we herein present a large number of studies performed in different countries, showing the most reliable DNA barcodes for species identification based on both mitochondrial (COI, cytb, 16S rDNA and 12S rDNA) and nuclear genes. Results are discussed considering the advantages and disadvantages of the different techniques in relation to different scientific issues. Special regard has been dedicated to a dual approach referring to both the consumer's health and the conservation of threatened species, with a special focus on the feasibility of the different genetic and genomic approaches in relation to both scientific objectives and permissible costs to obtain reliable traceability. [ABSTRACT FROM AUTHOR]

Ship hull fouling is recognized as an important vector for the introduction of nonindigenous species (NIS), which has been studied globally but no empirical works exist in the Southeast Pacific. The present study examined fouling organisms on the hulls of three ships in one international Chilean port, and compared them with those on settling plates and natural substrates. Also, genetic analyses were perfomed on the most common NIS recorded in order to explore number and potential origin of the genetic linages found. The highest number of total taxa and NIS were found on ship hulls, with three species identified as NIS in ship samplings. Settling plates displayed the largest number of taxa, with a large abundance of the invasive tunicate Ciona robusta, which also showed high mitochondrial genetic diversity. This study showed that Chilean coasts are subjected to both NIS colonization and propagule pressure through ship hulls. Biosecurity measures should urgently be taken on ship hulls along these coasts. [ABSTRACT FROM AUTHOR]

Background: Blood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources. Methods: We used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method. Results: Complete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively. Conclusions: Our findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel 'phylogenetic capture' methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens. [ABSTRACT FROM AUTHOR]

Globally, microplastics (MP) represent a growing burden for ecosystems due to their increasing presence at different trophic levels. In Ecuador, the lack of waste segregation has increased the quantity of waste, primarily organics and plastics, overloading landfills and water sources. Over time, plastics reduce in size and silently enter the food chain of animals, such as insects. The black soldier fly (BSF) larvae, Hermetia illucens (Linnaeus, 1758), is a species with devouring behavior used for waste management because of its beneficial qualities such as fly pest control, biomass production, and rapid organic waste degradation. Studies have uncovered the insect's ability to tolerate MP, and consider the possibility that they may be able to degrade polymers. For the first time in Ecuador, the present study characterized H. illucens using the sequences of different molecular markers. Finally, H. illucens' degrading capacity was evaluated in the presence of MP and decaying food residues, resembling landfill conditions. [ABSTRACT FROM AUTHOR]

Single-cell research has provided a breakthrough in biology to understand heterogeneous cell groups, such as tissues and organs, in development and disease. Molecular barcoding and subsequent sequencing technology insert a singlecell barcode into isolated single cells, allowing separation cell by cell. Given that multimodal information from a cell defines precise cellular states, recent technical advances in methods focus on simultaneously extracting multimodal data recorded in different biological materials (DNA, RNA, protein, etc.). This review summarizes recently developed singlecell multiomics approaches regarding genome, epigenome, and protein profiles with the transcriptome. In particular, we focus on how to anchor or tag molecules from a cell, improve throughputs with sample multiplexing, and record lineages, and we further discuss the future developments of the technology. [ABSTRACT FROM AUTHOR]

Agaricales species form pileate-stipitate fruiting bodies and play important roles in maintaining the terrestrial ecosystem as decomposers, symbionts, and pathogens. Approximately 23,000 Agaricales species have been known worldwide, and 937 species have been recorded in the Republic of Korea. However, most of them were identified solely based on morphological characteristics that often led to misidentifications. The specimens collected from 2018 to 2020 in the Republic of Korea were identified based on phylogenetic analysis of the internal transcribed spacer (ITS) sequences. Their identities were confirmed by microscopic characteristics. As a result, 14 Agaricales species were discovered for the first time in the Republic of Korea. They belonged to nine genera: Agaricus, Calocybe, Cortinarius, Hygrocybe, Inocybe, Lepista, Leucoagaricus, Marasmius, and Psathyrella. Detailed macroscopic and microscopic descriptions were provided to help distinguish these species. The morphological and molecular data provided in this study will serve as reliable references for the identification of Agaricales species.

Globally, microplastics (MP) represent a growing burden for ecosystems due to their increasing presence at different trophic levels. In Ecuador, the lack of waste segregation has increased the quantity of waste, primarily organics and plastics, overloading landfills and water sources. Over time, plastics reduce in size and silently enter the food chain of animals, such as insects. The black soldier fly (BSF) larvae, Hermetia illucens (Linnaeus, 1758), is a species with devouring behavior used for waste management because of its beneficial qualities such as fly pest control, biomass production, and rapid organic waste degradation. Studies have uncovered the insect’s ability to tolerate MP, and consider the possibility that they may be able to degrade polymers. For the first time in Ecuador, the present study characterized H. illucens using the sequences of different molecular markers. Finally, H. illucens’ degrading capacity was evaluated in the presence of MP and decaying food residues, resembling landfill conditions.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing >50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Agaricales species form pileate-stipitate fruiting bodies and play important roles in maintaining the terrestrial ecosystem as decomposers, symbionts, and pathogens. Approximately 23,000 Agaricales species have been known worldwide, and 937 species have been recorded in the Republic of Korea. However, most of them were identified solely based on morphological characteristics that often led to misidentifications. The specimens collected from 2018 to 2020 in the Republic of Korea were identified based on phylogenetic analysis of the internal transcribed spacer (ITS) sequences. Their identities were confirmed by microscopic characteristics. As a result, 14 Agaricales species were discovered for the first time in the Republic of Korea. They belonged to nine genera: Agaricus, Calocybe, Cortinarius, Hygrocybe, Inocybe, Lepista, Leucoagaricus, Marasmius, and Psathyrella. Detailed macroscopic and microscopic descriptions were provided to help distinguish these species. The morphological and molecular data provided in this study will serve as reliable references for the identification of Agaricales species. [ABSTRACT FROM AUTHOR]

Abstract Background The ixodid tick genera Rhipicephalus and Haemaphysalis contain several species of medical and/or veterinary importance, but their diversity in some regions of the world remains under-explored. For instance, very few modern studies have been performed on the taxonomy of these genera on the Arabian Peninsula. Methods In this study, we trapped small mammals in the 'Asir Mountains of south-western Saudi Arabia and collected tick specimens for morphological examination and molecular barcoding, targeting three mitochondrial loci: cox1, 16S rRNA and 12S rRNA. Results We obtained a total of 733 ticks (608 Haemaphysalis spp. and 125 Rhipicephalus spp.) from 75 small mammal hosts belonging to six species. All tick specimens were immature except for nine adults recovered from a hedgehog (Paraechinus aethiopicus). Morphologically, the Rhipicephalus ticks resembled R. camicasi, but the Haemaphysalis ticks showed differences in palp morphology compared with species previously described from Saudi Arabia. Phylogenetic analysis and automatic barcode gap discovery identified a novel clade of Rhipicephalus sp. representing most of the nymphs. This was most closely related to R. leporis, R. guilhoni and R. linnaei. The adult ticks and a small proportion of nymphs clustered with R. camicasi sequences from a previous study. Finally, the Haemaphysalis nymphs formed two distinct clades that were clearly separated from all reference sequences but closest to some African species. Conclusions This apparent high level of tick diversity observed in a single study site of only ~ 170 km2, on a relatively small number of hosts, highlights the potential for the discovery of new tick species on the Arabian Peninsula. Graphical Abstract

The recent increase in international fish trade leads to the need for improving the traceability of fishery products. In relation to this, consistent monitoring of the production chain focusing on technological developments, handling, processing and distribution via global networks is necessary. Molecular barcoding has therefore been suggested as the gold standard in seafood species traceability and labelling. This review describes the DNA barcoding methodology for preventing food fraud and adulteration in fish. In particular, attention has been focused on the application of molecular techniques to determine the identity and authenticity of fish products, to discriminate the presence of different species in processed seafood and to characterize raw materials undergoing food industry processes. In this regard, we herein present a large number of studies performed in different countries, showing the most reliable DNA barcodes for species identification based on both mitochondrial (COI, cytb, 16S rDNA and 12S rDNA) and nuclear genes. Results are discussed considering the advantages and disadvantages of the different techniques in relation to different scientific issues. Special regard has been dedicated to a dual approach referring to both the consumer’s health and the conservation of threatened species, with a special focus on the feasibility of the different genetic and genomic approaches in relation to both scientific objectives and permissible costs to obtain reliable traceability.

Six species of Sticta are described as new to science on the basis of material from Bolivia and supported by phylogenetic analysis of the fungal ITS barcoding marker. The species were resolved in all three of the clades (I, II, III) widespread and common in the Neotropics, as defined in an earlier study on the genus. Comparison with material from neighbouring countries (i.e. Colombia, Ecuador, Peru) suggests that these new species may be potentially endemic to the Bolivian Yungas ecoregion. For each species, a detailed morphological and anatomical description is given. Sticta amboroensis Ossowska, Kukwa, B. Moncada & Lücking is a medium-sized green-algal species with laminal to submarginal apothecia with hirsute margins and with light to dark brown lower tomentum. Sticta aymara Ossowska, Kukwa, B. Moncada, Flakus, Rodriguez-Flakus & Lücking is a comparatively small cyanobacterial taxon with Nostoc as photo-biont, laminal, richly branched, aggregate isidia and a golden to chocolate-brown lower tomentum. The medium-sized, cyanobacterial S. bicellulata Ossowska, Kukwa, B. Moncada & Lücking has cyanobacterial photobiont, bicellular ascospores, apothecia with white to golden-brown hairs on the margins, K+ violet apothecial margin (ring around disc) and epihymenium and a white to dark brown lower tomentum. In contrast, the green-algal species, S. carrascoensis Ossowska, Kukwa, B. Moncada & Lücking is characterised by its large size, apothecia with dark brown hairs on the margins and a yellow medulla. The cyanobacterial S. catharinae Ossowska, B. Moncada, Kukwa, Flakus, Rodriguez-Flakus & Lücking forms stipitate thalli with Nostoc as photobiont, abundant, laminal to submarginal apothecia and a golden-brown lower tomentum. Finally, the cyanobacterial S. pseudoimpressula Ossowska, Kukwa, B. Moncada & Lücking produces laminal apothecia with an orange-yellow line of pruina along the margins which reacts K+ carmine-red. In addition to the six new Bolivian taxa, the cyanobacterial S. narinioana B. Moncada, Ossowska & Lücking is described as new from Colombia and it represents the closely-related sister species of the Bolivian S. aymara; it differs from the latter largely in the marginal instead of laminal isidia. [ABSTRACT FROM AUTHOR]

Abstract Background Rodent borne hantaviruses are emerging viruses infecting humans through inhalation. They cause hemorrhagic fever with renal syndrome and hemorrhagic cardiopulmonary syndrome. Recently, hantaviruses have been detected in other small mammals such as Soricomorpha (shrews, moles) and Chiroptera (bats), suggested as reservoirs for potential pandemic viruses and to play a role in the evolution of hantaviruses. It is important to study the global virome in different reservoirs, therefore our aim was to investigate whether shrews in Sweden carried any hantaviruses. Moreover, to accurately determine the host species, we developed a molecular method for identification of shrews. Method Shrews (n = 198), caught during 1998 in Sweden, were screened with a pan-hantavirus PCR using primers from a conserved region of the large genome segment. In addition to morphological typing of shrews, we developed a molecular based typing method using sequencing of the mitochondrial cytochrome C oxidase I (COI) and cytochrome B (CytB) genes. PCR amplified hantavirus and shrew fragments were sequenced and phylogenetically analysed. Results Hantavirus RNA was detected in three shrews. Sequencing identified the virus as Seewis hantavirus (SWSV), most closely related to previous isolates from Finland and Russia. All three SWSV sequences were retrieved from common shrews (Sorex araneus) sampled in Västerbotten County, Sweden. The genetic assay for shrew identification was able to identify native Swedish shrew species, and the genetic typing of the Swedish common shrews revealed that they were most similar to common shrews from Russia. Conclusion We detected SWSV RNA in Swedish common shrew samples and developed a genetic assay for shrew identification based on the COI and CytB genes. This was the first report of presence of hantavirus in Swedish shrews.

Plasma circulating tumor DNA (ctDNA) analysis has emerged as a minimally invasive means to perform molecular tumor typing. Here we developed a custom ultra-sensitive ctDNA next-generation sequencing assay using molecular barcoding technology and off-the-shelf reagents combined with bioinformatics tools for enhanced ctDNA analysis. Assay performance was assessed via a spike-in experiment and the technique was applied to analyze 41 plasma samples from men with advanced prostate cancer. Orthogonal validation was performed using a commercial assay. Sensitivity and specificity of 93 and 99.5% were recorded for ultra-rare somatic variants (

Background: The ixodid tick genera Rhipicephalus and Haemaphysalis contain several species of medical and/or veterinary importance, but their diversity in some regions of the world remains under-explored. For instance, very few modern studies have been performed on the taxonomy of these genera on the Arabian Peninsula. Methods: In this study, we trapped small mammals in the 'Asir Mountains of south-western Saudi Arabia and collected tick specimens for morphological examination and molecular barcoding, targeting three mitochondrial loci: cox1, 16S rRNA and 12S rRNA. Results: We obtained a total of 733 ticks (608 Haemaphysalis spp. and 125 Rhipicephalus spp.) from 75 small mammal hosts belonging to six species. All tick specimens were immature except for nine adults recovered from a hedgehog (Paraechinus aethiopicus). Morphologically, the Rhipicephalus ticks resembled R. camicasi, but the Haemaphysalis ticks showed differences in palp morphology compared with species previously described from Saudi Arabia. Phylogenetic analysis and automatic barcode gap discovery identified a novel clade of Rhipicephalus sp. representing most of the nymphs. This was most closely related to R. leporis, R. guilhoni and R. linnaei. The adult ticks and a small proportion of nymphs clustered with R. camicasi sequences from a previous study. Finally, the Haemaphysalis nymphs formed two distinct clades that were clearly separated from all reference sequences but closest to some African species. Conclusions: This apparent high level of tick diversity observed in a single study site of only ~ 170 km2, on a relatively small number of hosts, highlights the potential for the discovery of new tick species on the Arabian Peninsula. [ABSTRACT FROM AUTHOR]

[Display omitted] • Immunotherapies have demonstrated long-term and durable responses in NSCLC patients, although they appear to be effective in a subset of patients. • Predictive biomarkers are needed to identify those likely to achieve the greatest benefit. • Advances in spatial profiling methodologies, which combine genomic/proteomic data with sequential imaging is likely to lead to deep insights into the underlying tumour tissue biology. • Spatial transcriptomics has enabled a fundamental understanding of the tumour and immune cell interactions. • The field of spatial transcriptomics is likely to identify prognostic cellular profiles, locations, interactions and signatures. Non-small cell lung cancer (NSCLC) is one of the most common cancers globally and has a 5-year survival rate ~20%. Immunotherapies have demonstrated long-term and durable responses in NSCLC patients, although they appear to be effective in only a subset of patients. A more comprehensive understanding of the underlying tumour biology may contribute to identifying those patients likely to achieve optimal outcomes. Profiling the tumour microenvironment (TME) has shown to be beneficial in addressing fundamental tumour-immune cell interactions. Advances in multiplexing immunohistochemistry and molecular barcoding has led to recent advances in profiling genes and proteins in NSCLC. Here, we review the recent advancements in spatial profiling technologies for the analysis of NSCLC tissue samples to gain new insights and therapeutic options for NSCLC. The combination of spatial transcriptomics combined with advanced imaging is likely to lead to deep insights into NSCLC tissue biology, which can be a powerful tool to predict likelihood of response to therapy. [ABSTRACT FROM AUTHOR]

Abstract Background In lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations. Methods We performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh–frozen lung cancer samples. Results Profiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018). Conclusions Our findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities.

Background: Hepatocellular carcinoma (HCC) is the second-most-common cause of cancer- related deaths worldwide, and an accurate and non-invasive biomarker for the early detection and monitoring of HCC is required. We assessed pathogenic variants of HCC driver genes in cell-free DNA (cfDNA) from HCC patients who had not undergone systemic therapy. Methods: Plasma cfDNA was collected from 20 HCC patients, and deep sequencing was performed using a customized cfDNA next-generation sequencing panel, targeting the major HCC driver genes (TP53, CTNNB1, TERT) that incorporates molecular barcoding. Results: In 13/20 (65%) patients, we identified at least one pathogenic variant of two major HCC driver genes (TP53 and CTNNB1), including 16 variants of TP53 and nine variants of CTNNB1. The TP53 and CTNNB1 variants showed low allele frequencies, with median values of 0.17% (range: 0.06%--6.99%) and 0.07% (range: 0.05%--0.96%), respectively. However, the molecular coverage of variants was sufficient, with median values of 5,543 (range: 2,317--9,088) and 7,568 (range: 2,400--9,633) for TP53 and CTNNB1 variants, respectively. Conclusions: Our targeted DNA sequencing successfully identified low-frequency pathogenic variants in the cfDNA from HCC patients by achieving high coverage of unique molecular families. Our results support the utility of cfDNA analysis to identify somatic gene variants in HCC patients. [ABSTRACT FROM AUTHOR]

Background: Rodent borne hantaviruses are emerging viruses infecting humans through inhalation. They cause hemorrhagic fever with renal syndrome and hemorrhagic cardiopulmonary syndrome. Recently, hantaviruses have been detected in other small mammals such as Soricomorpha (shrews, moles) and Chiroptera (bats), suggested as reservoirs for potential pandemic viruses and to play a role in the evolution of hantaviruses. It is important to study the global virome in different reservoirs, therefore our aim was to investigate whether shrews in Sweden carried any hantaviruses. Moreover, to accurately determine the host species, we developed a molecular method for identification of shrews. Method: Shrews (n = 198), caught during 1998 in Sweden, were screened with a pan-hantavirus PCR using primers from a conserved region of the large genome segment. In addition to morphological typing of shrews, we developed a molecular based typing method using sequencing of the mitochondrial cytochrome C oxidase I (COI) and cytochrome B (CytB) genes. PCR amplified hantavirus and shrew fragments were sequenced and phylogenetically analysed. Results: Hantavirus RNA was detected in three shrews. Sequencing identified the virus as Seewis hantavirus (SWSV), most closely related to previous isolates from Finland and Russia. All three SWSV sequences were retrieved from common shrews (Sorex araneus) sampled in Västerbotten County, Sweden. The genetic assay for shrew identification was able to identify native Swedish shrew species, and the genetic typing of the Swedish common shrews revealed that they were most similar to common shrews from Russia. Conclusion: We detected SWSV RNA in Swedish common shrew samples and developed a genetic assay for shrew identification based on the COI and CytB genes. This was the first report of presence of hantavirus in Swedish shrews. [ABSTRACT FROM AUTHOR]

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Eragrostis tef is an important cereal crop in Ethiopia with excellent storage properties, high–quality food, and the unique ability to thrive in extreme environmental conditions. However, the application of advanced molecular tools for breeding and conservation of these species is extremely limited. Therefore, developing chloroplast genome resources and high-resolution molecular markers are valuable to E. tef population and biogeographic studies. In the current study, we assembled and compared the complete plastomes of 32 E. tef accessions. The size of the plastomes ranged from 134,349 to 134,437 bp with similar GC content (∼38.3%). Genomes annotations revealed 112 individual genes, including 77 protein-coding, 31 tRNA, and 4 rRNA genes. Comparison of E. tef plastomes revealed a low degree of intraspecific sequence variations and no structural differentiations. Furthermore, we found 34 polymorphic sites (13 cpSSRs, 12 InDels, and 9 SNPs) that can be used as valuable DNA barcodes. Among them, the majority (88%) of the polymorphic sites were identified in the noncoding genomic regions. Nonsynonymous (ka) and synonymous (ks) substitution analysis showed that all PCGs were under purifying selection (ka/ks

Coffee is a popular beverage in the world and a valuable agricultural export commodity for several developing countries. Coffea arabica is one of the widely cultivated species and is believed to have originated from Ethiopia. Currently, a large number of C. arabica accessions are conserved ex situ in field gene banks in Ethiopia. However, there is no useful molecular barcoding to identify the conserved accessions, which presents a challenge to sustainable utilization of this economic species. To identify polymorphic chloroplast markers for the use in various studies, the complete chloroplast genome sequences of 24 C. arabica accessions were assembled and screened for variable regions in this study. The total length of the individual chloroplast genome sequence varied from 155,059 to 155,192 bp. Genome annotation revealed a total of 114 unique genes, consisting of 80 protein-coding, 30 tRNA, and four rRNA genes in all the 24 genomes. Twenty-four polymorphic regions were identified and developed. Most (71%) of the variable regions were found in non-coding sequence, and 54% of the variations detected are single base pairs. Three non-synonymous SNPs were found in accD, rpoA, and ycf1 genes. The markers developed were able to cluster the majority of the coffee accessions according to their geographical regions of collection. Five out of 24 newly developed markers were validated by assessing the diversity levels that were assessed in five original regions. The southwest region displayed the highest genetic diversity (HE = 0.224; I = 0.370) and might become a potential coffee genetic resource bank. Overall, the polymorphic markers developed and validated in this study could be a useful genomic resource for molecular breeding, identification, and biogeography studies of this commercially important crop. [ABSTRACT FROM AUTHOR]

The Red Sea intertidal gastropod Nerita sanguinolenta is reported here for the first time from the Northern Mediterranean coast of Israel, where it was found at the landward face of a vermetid reef on 11 November 2019, and identified morphologically and by using molecular barcoding. Possible vectors for its introduction primarily include ship ballast or larval transport via the Suez Canal in multiple migration events. Investigation of the thermal affinities of N. sanguinolenta is required for understanding the potential for its proliferation in the Mediterranean Sea. [ABSTRACT FROM AUTHOR]

Tumor heterogeneity is a hallmark of many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), and an inherent consequence of the clonal evolution of cancers. As such, it is considered the underlying concept of many characteristics of the disease, including the ability to metastasize, adapt to different microenvironments, and to develop therapy resistance. Undoubtedly, the high mortality of PDAC can be attributed to a high extent to these properties. Despite its apparent importance, studying tumor heterogeneity has been a challenging task, mainly due to its complexity and lack of appropriate methods. However, in recent years molecular DNA barcoding has emerged as a sophisticated tool that allows mapping of individual cells or subpopulations in a cell pool to study heterogeneity and thus devise new personalized treatment strategies. In this review, we provide an overview of genetic and non-genetic inter- and intra-tumor heterogeneity and its impact on (personalized) treatment strategies in PDAC and address how DNA barcoding technologies work and can be applied to study this clinically highly relevant question.

Abstract Background RNA-Seq measures gene expression by counting sequence reads belonging to unique cDNA fragments. Molecular barcodes commonly in the form of random nucleotides were recently introduced to improve gene expression measures by detecting amplification duplicates, but are susceptible to errors generated during PCR and sequencing. This results in false positive counts, leading to inaccurate transcriptome quantification especially at low input and single-cell RNA amounts where the total number of molecules present is minuscule. To address this issue, we demonstrated the systematic identification of molecular species using transposable error-correcting barcodes that are exponentially expanded to tens of billions of unique labels. Results We experimentally showed random-mer molecular barcodes suffer from substantial and persistent errors that are difficult to resolve. To assess our method’s performance, we applied it to the analysis of known reference RNA standards. By including an inline random-mer molecular barcode, we systematically characterized the presence of sequence errors in random-mer molecular barcodes. We observed that such errors are extensive and become more dominant at low input amounts. Conclusions We described the first study to use transposable molecular barcodes and its use for studying random-mer molecular barcode errors. Extensive errors found in random-mer molecular barcodes may warrant the use of error correcting barcodes for transcriptome analysis as input amounts decrease.

Hydrozoa medusae undergo blooms and seasonal fluctuations; however the drivers of such fluctuations are unknown. To understand how medusa populations fluctuate in response to seasonal factors such as temperature, salinity, dissolved oxygen, and chlorophyll a, and to enhance our taxonomic knowledge of Hydrozoa in Galveston Bay (TX), we performed frequent plankton sampling from September 2015 to September 2016. We collected 1,321 medusae in 190 sampling days. Using molecular barcoding and morphological analyses we identified 25 species, of which 21 are a first record for Galveston Bay and eight for the Gulf of Mexico. Daily medusa abundance is non-linearly related to temperature, with peak abundance estimated with multivariate regression analysis at approximately 21C. The role that temperature plays in driving medusa abundance has implications for future climate change scenarios, given that temperature in the Gulf of Mexico is expected to rise 4 °C by the end of the century. We also show that the biodiversity of the Galveston Bay and the Gulf of Mexico is underestimated and that molecular barcoding is an important and efficient tool to identify large number of medusae. We conclude that dense plankton sampling is necessary to capture both diversity and abundance of planktonic medusae. [ABSTRACT FROM AUTHOR]

Using a ~ 600 bps fragment of the mitochondrial 16S gene, we reconstruct the phylogenetic history of the species complex Pennaria disticha and of the genus Turritopsis. P. disticha and, within the genus Turritopsis, Turritopsis dohrnii are considered invasive species with a very broad geographical range. We show that the invasive T. dohrnii has reached the Gulf of Mexico (GoM) and Brazil. We heavily sampled the Bocas del Toro region, Panama, where three species of Turritopsis (the invasive T. dohrnii and the endemic T. sp. 1 and T. sp. 4) have been recorded, and found that the invasive T. dohrnii is the most common species, while the endemic species were either very rare (T. sp. 1) or were not found (T. sp. 4). We show that T. dohrnii from Bocas del Toro can revert its life cycle from medusa to polyp, while T. sp. 1 may lack this capability. Finally, we find that in the Maldives, Emirates, and the Red Sea, there are morphologically and genetically distinct Turritopsis lineages. With regard to Pennaria disticha, we analyzed new sequences from Thuwal (Saudi Arabia, Red Sea), Sint Eustatius (the Netherlands, Caribbean), Bocas del Toro (Panama, Caribbean Sea), Bergeggi (Italy, Mediterranean Sea), Tricase (Italy, Mediterranean Sea), Dibba Al-Fujairah (United Arab Emirates, Gulf of Oman), and Galveston (Texas, Gulf of Mexico). We confirm the presence of several clades and putative cryptic species that indicates multiple invasive episodes and undercover a new clade from the Gulf of Oman. Finally, species delimitation analyses recover 2 to 3 clades as separate species within P. disticha, and 7 to 9 species within Turritopsis, with T. lata being a complex of 2–4 cryptic species. [ABSTRACT FROM AUTHOR]

Background: In lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations.Methods: We performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh-frozen lung cancer samples.Results: Profiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018).Conclusions: Our findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities. [ABSTRACT FROM AUTHOR]

Abstract In this study, we examine the current pattern of tick diversity and host use in French Guiana, South America, from 97 sampling localities encompassing peri-urban, rural and natural habitats. We collected 3395 ticks, including 1485 specimens from 45 vertebrate species (humans, domestic and wild animals) and 1910 questing specimens from vegetation. Morphological examinations identified 22 species belonging to six genera: Amblyomma (16 species), Rhipicephalus (two species), Ixodes (one species), Dermacentor (one species), Haemaphysalis (one species), Ornithodoros (one species). To facilitate future identification, we produced a bank of pictures of different stages for all these species. Taxonomic identification was then confirmed by molecular characterization of two mitochondrial genes, cytochrome c oxidase CO1 and 16S rDNA. Eleven of the 22 reported species were collected on humans, six on domestic animals and 12 on wild animals. The most widespread tick species collected were A. cajennense sensu stricto and, to a lesser extent, A. oblongoguttatum ; both of these species were frequently found on humans. We used these results to discuss the tick-associated risks for human and animal health in French Guiana. [ABSTRACT FROM AUTHOR]

DNA barcoding has become an important aspect in biodiversity research, but is ineffective without comprehensive databases that can relate molecular sequences to scientific names. Hermit crabs, or paguroids, are an important component of the marine benthos, and are vastly understudied in southern Africa, a globally important bioregion. The study provides 194 cytochrome c oxidase subunit I (COI) barcode sequences (> 550 bp) of 43 nominal and 12 putative species from 19 genera and four families of Paguroidea. Preliminary COI-based phylogenetic analysis suggested some overall pattern of monophyly in the Parapaguridae and Paguristes Dana, 1851 and Dardanus Paulˊson, 1875. Large groups in Diogenidae, however, such as Calcinus Dana, 1851 and Diogenes Dana, 1851, were recovered as poly- or paraphyletic. Testing the dataset for barcoding efficacy revealed that the technique can reach identification rates near 100% success. A barcoding gap was apparent for almost all but one problematic taxon. The optimum threshold range to discriminate between species for this set of data was calculated at 3.7–3.9%, although the overall mean intraspecific distance was well below 1%. Furthermore, glaucothoe larvae of two deep-water paguroids were matched to names in the new molecular library. The COI marker alone, however, did not provide clear species identifications in three problematic species, Areopaguristes cf. jousseaumei (Bouvier, 1892), Goreopagurus poorei Lemaitre & McLaughlin, 2003, and Pagurus cuanensis Bell, 1846. Despite the dataset having considerable species coverage of 55 total taxa, it is still estimated to be only about 50% complete in terms of the South African fauna. Future work should therefore increase coverage to other species in South Africa and expand geographic coverage for widely distributed species. [ABSTRACT FROM AUTHOR]

Understanding vertebrate–vector interactions is vitally important for understanding the transmission dynamics of arthropod-vectored pathogens and depends on the ability to accurately identify the vertebrate source of blood-engorged arthropods in field collections using molecular methods. A decade ago, molecular techniques being applied to arthropod blood meal identification were thoroughly reviewed, but there have been significant advancements in the techniques and technologies available since that time. This review highlights the available diagnostic markers in mitochondrial and nuclear DNA and discusses their benefits and shortcomings for use in molecular identification assays. Advances in real-time PCR, high resolution melting analysis, digital PCR, next generation sequencing, microsphere assays, mass spectrometry, and stable isotope analysis each offer novel approaches and advantages to bloodmeal analysis that have gained traction in the field. New, field-forward technologies and platforms have also come into use that offer promising solutions for point-of-care and remote field deployment for rapid bloodmeal source identification. Some of the lessons learned over the last decade, particularly in the fields of DNA barcoding and sequence analysis, are discussed. Though many advancements have been made, technical challenges remain concerning the prevention of sample degradation both by the arthropod before the sample has been obtained and during storage. This review provides a roadmap and guide for those considering modern techniques for arthropod bloodmeal identification and reviews how advances in molecular technology over the past decade have been applied in this unique biomedical context.

Microbes are sensitive indicators of estuarine processes because they respond rapidly to dynamic disturbance events. As most of the world's population lives in urban areas and climate change-related disturbance events are becoming more frequent, estuaries bounded by cities are experiencing increasing stressors, at the same time that their ecosystem services are required more than ever. Here, using a multidisciplinary approach, we determined the response of planktonic microbial assemblages in response to seasonality and a rainfall disturbance in an urban estuary bounded by Australia's largest city, Sydney. We used molecular barcoding (16S, 18S V4 rRNA) and microscopy-based identification to compare microbial assemblages at locations with differing characteristics and urbanisation histories. Across 142 samples, we identified 8,496 unique free-living bacterial zOTUs, 8,175 unique particle associated bacterial zOTUs, and 1,920 unique microbial eukaryotic zOTUs. Using microscopy, we identified only the top <10% abundant, larger eukaryotic taxa (>10 µm), however quantification was possible. The site with the greater history of anthropogenic impact showed a more even community of associated bacteria and eukaryotes, and a significant increase in dissolved inorganic nitrogen following rainfall, when compared to the more buffered site. This coincided with a reduced proportional abundance of Actinomarina and Synechococcus spp., a change in SAR 11 clades, and an increase in the eukaryotic microbial groups Dinophyceae, Mediophyceae and Bathyoccocaceae, including a temporary dominance of the harmful algal bloom dinoflagellate Prorocentrum cordatum (syn. P. minimum). Finally, a validated hydrodynamic model of the estuary supported these results, showing that the more highly urbanised and upstream location consistently experienced a higher magnitude of salinity reduction in response to rainfall events during the study period. The best abiotic variables to explain community dissimilarities between locations were TDP, PN, modelled temperature and salinity (r = 0.73) for the free living bacteria, TP for the associated bacteria (r = 0.43), and modelled temperature (r = 0.28) for the microbial eukaryotic communities. Overall, these results show that a minor disturbance such as a brief rainfall event can significantly shift the microbial assemblage of an anthropogenically impacted area within an urban estuary to a greater degree than a seasonal change, but may result in a lesser response to the same disturbance at a buffered, more oceanic influenced location. Fine scale research into the factors driving the response of microbial communities in urban estuaries to climate related disturbances will be necessary to understand and implement changes to maintain future estuarine ecosystem services. [Display omitted] [ABSTRACT FROM AUTHOR]

• Traditional technologies are insufficient for low-frequency mutation detection. • Multiple loci are necessary for higher sensitivity in ctDNA study. • Noise reduction have become indispensable for detection of ctDNA mutations. • Liquid biopsy needs to take a step into clinical setting through clinical trials. Cancer has become a global health issue and liquid biopsy has emerged as a non-invasive tool for various applications. In cancer, circulating tumor DNA (ctDNA) can be detected from cell-free DNA (cfDNA) obtained from plasma and has potential for early diagnosis, treatment, resistance, minimal residual disease detection, and tumoral heterogeneity identification. However, the low frequency of ctDNA requires techniques for accurate analysis. Multitarget assay such as Next Generation Sequencing (NGS) need improvement to achieve limits of detection that can identify the low frequency variants present in the cfDNA. In this review, we provide a general overview of the use of cfDNA and ctDNA in cancer, and discuss techniques developed to optimize NGS as a tool for ctDNA detection. We also summarize the results obtained using NGS strategies in both investigational and clinical contexts. [ABSTRACT FROM AUTHOR]

Thymomas are rare mediastinal tumors that are difficult to treat and pose a major public health concern. Identifying mutations in target genes is vital for the development of novel therapeutic strategies. Type A thymomas possess a missense mutation in GTF2I (chromosome 7 c.74146970T>A) with high frequency. However, the molecular pathways underlying the tumorigenesis of other thymomas remain to be elucidated. We aimed to detect this missense mutation in GTF2I in other thymoma subtypes (types B). This study involved 22 patients who underwent surgery for thymomas between January 2014 and August 2019. We isolated tumor cells from formalin-fixed paraffin-embedded tissues from the primary lesions using laser-capture microdissection. Subsequently, we performed targeted sequencing to detect mutant GTF2I coupled with molecular barcoding. We used PyClone analysis to determine the fraction of tumor cells harboring mutant GTF2I. We detected the missense mutation (chromosome 7 c.74146970T>A) in GTF2I in 14 thymomas among the 22 samples (64%). This mutation was harbored in many type B thymomas as well as type A and AB thymomas. The allele fraction for the tumors containing the mutations was variable, primarily owing to the coexistence of normal lymphocytes in the tumors, especially in type B thymomas. PyClone analysis revealed a high cellular prevalence of mutant GTF2I in tumor cells. Mutant GTF2I was not detected in other carcinomas (lung, gastric, colorectal, or hepatocellular carcinoma) or lymphomas. In conclusion, the majority of thymomas harbor mutations in GTF2I that can be potentially used as a novel therapeutic target in patients with thymomas.