Molecular Barcoding Research Articles

First insight into molecular diversity and DNA barcode library of epikarst-dwelling invertebrates in the Western Carpathians

DNA barcoding represents a handy tool for species identification. In addition, it serves as a complementary approach that improves the characterisation of evolutionary lineages and facilitates the detection of potentially undescribed and cryptic species. Based on the case study in the Western Carpathians, which belong to the Carpathian biodiversity hotspot, we have compiled the first DNA barcode reference library for molecular identification of invertebrates associated with epikarst, a unique, yet understudied, shallow subterranean aquatic habitat that extends at the interface between the soil and carbonate rocks. We analysed invertebrates collected in 2019-2020 from epikarst water that continuously seeps into four caves of the Demänovský Cave System in northern Slovakia. The standard barcode marker of the mitochondrial COI gene was amplified in more than 920 individuals of aquatic, semi-aquatic, and terrestrial invertebrates. The final data set consisted of 784 barcode sequences representing 36 morphospecies, the majority (98.3%) belonging to Arthropoda. Automated cluster delineation using the Barcode of Life Data System (BOLD) revealed 60 Barcode Index Numbers (BINs), of which 43 BINs were new to BOLD, representing mostly typical subterranean species. Almost 20% of the morphospecies displayed high intraspecific variation (>2.2%), suggesting the need for further investigation to assess potential taxonomic problems or cryptic diversity. Our results also indicated the existence of several yet undescribed invertebrate species and possible heteroplasmy or COI numts in the collembolan Megalothorax sp. (incertus species group). The resulting DNA barcode library represents a significant advance not only in the characterisation of epikarst biodiversity but also in the understanding of subterranean biodiversity in general, paving the way for future complex evolutionary and biogeographical studies.

Bacterial Species Associate on the Body Surface of Musca domestica L from Various Habitats based on 16S rRNA Sequencing

This study aims to identify bacteria isolated from the body surface of house flies from various habitats using 16S rRNA molecular barcodes. Houseflies were isolated from forests, hospitals, traditional markets, modern markets and landfills. 25 house flies isolated in each habitat. House flies were preserved in sterile bags. Bacterial isolation was carried out using nutrient agar media in 100 mm Petri dishes. The isolates obtained were pure cultured until a single isolate was obtained. Single isolates were extracted using Geneaid’s Presto TM Mini gDNA Bacteria Kit. The extracted bacterial total DNA was used as a template for amplification using primer 16s rRNA gene by PCR method. Nucleotide sequencing uses Singapore’s First BASE sequencing service. The results showed that single-house fly isolates from the Fish Auction (P.L.) showed a 99.11% similarity with Sphingobacterium faecium [CP094931.1]. Traditional market bacterial isolates (P.T.) showed 97% similarity with Pseudochrobactrum sp. XF203. Hospital bacterial isolates (R.S.) showed 99.11% similarity with S. faecium [CP094931.1]. Bacterial isolates from residential areas (PM) showed 99% similarity with Brucella abortus RB51-AHVLA. Bacterial isolates from the forest (H.T.) showed 94% similarity with Bacillus paralicheniformis [CP043501.1]. There are associated bacteria that are used as biotechnology agents. Exploration of bacteria and even microbes associated with M. domestica is still extensive to be studied in the future.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

Distinct neuroinflammatory signatures exist across genetic and sporadic amyotrophic lateral sclerosis cohorts.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. ALS is on a pathogenetic disease spectrum with frontotemporal dementia, referred to as ALS-frontotemporal spectrum disorder (ALS-FTSD). For mutations associated with ALS-FTSD, such as the C9orf72 hexanucleotide repeat expansion, the molecular factors associated with heterogeneity along this spectrum require further characterization. Here, using a targeted NanoString molecular barcoding approach, we interrogate neuroinflammatory dysregulation and heterogeneity at the level of gene expression in post-mortem motor cortex tissue from a cohort of clinically heterogeneous C9-ALS-FTSD cases. We identified 20 dysregulated genes in C9-ALS-FTSD, with enrichment of microglial and inflammatory response gene sets. Two genes with significant correlations to available clinical metrics were selected for validation: FKBP5, a correlate of cognitive function, and brain-derived neurotrophic factor (BDNF), a correlate of disease duration. FKBP5 and its signalling partner, NF-κB, appeared to have a cell type-specific staining distribution, with activated (i.e. nuclear) NF-κB immunoreactivity in C9-ALS-FTSD. Expression of BDNF, a correlate of disease duration, was confirmed to be higher in individuals with long compared to short disease duration using BaseScope™ in situ hybridization. Our analyses also revealed two distinct neuroinflammatory panel signatures (NPS), NPS1 and NPS2, delineated by the direction of expression of proinflammatory, axonal transport and synaptic signalling pathways. We compared NPS between C9-ALS-FTSD cases and those from sporadic ALS and SOD1-ALS cohorts and identified NPS1 and NPS2 across all cohorts. Moreover, a subset of NPS was also able to separate publicly available RNA sequencing data from independent C9-ALS and sporadic ALS cohorts into two inflammatory subgroups. Importantly, NPS subgroups did not clearly segregate with available demographic, genetic, clinical or pathological features, highlighting the value of molecular stratification in clinical trials for inflammatory subgroup identification. Our findings thus underscore the importance of tailoring therapeutic approaches based on distinct molecular signatures that exist between and within ALS-FTSD cohorts.

A versatile CRISPR-based system for lineage tracing in living plants.

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.

Intragenomic diversity of the V9 hypervariable domain in eukaryotes has little effect on metabarcoding

Metabarcoding revolutionized our understanding of diversity and ecology of microorganisms in different habitats. However, it is also associated with several inherent biases, one of which is associated with intragenomic diversity of a molecular barcode. Here, we compare intragenomic variability of the V9 region of the 18S rRNA gene in 19 eukaryotic phyla abundant in marine plankton. The level of intragenomic variability is comparable across all the phyla, and in most genomes and transcriptomes one V9 sequence and one OTU is predominant. However, most of the variability observed at the barcode level is probably caused by sequencing errors and is mitigated by using a denoising tool, DADA2. The SWARM algorithm commonly used in metabarcoding studies is not optimal for collapsing genuine and erroneous sequences into a single OTU, leading to anoverestimation of diversity in metabarcoding data. For an unknown reason,SWARM inflates diversity of eupelagonemids more than that of other eukaryotes.

Morphological and Molecular Barcoding Characterization of Four Invasive Species of Solanaceae in the Southern Region of Saudi Arabia

Invasive plants are the second greatest threat to plant diversity, after habitat loss due to human activities. Plant diversity hotspots and islands, as well as farmlands, woodlands, forests, grasslands, and populated areas, are all threatened by invasive species. Over the centuries, and especially in the last few decades, trade and travel have increased dramatically, hastening the introduction of alien species. Although comprehensive sampling is necessary for DNA barcoding, it is rarely done due to a lack of materials. In practice, only a few species from each genus must be identified, rather than all of them. In many of Saudi Arabia's most problematic habitats in the southwestern region, where the presence of invasive species was significant. As a result, in the current application of DNA barcodes, identifying a given species using a limited sample is important. In the case of incomplete sampling, DNA barcodes can provide reliable specific-species discrimination. For that purpose, the current study was designed to identify the four invasive species in the southern part of Saudi Arabia using morphological and 4 molecular barcoding (ITS, PsbA, rbcla, matk,) markers were used for the identification and characterization of invasive species. The results suggested identifying the 4 invasive Solanaceae plants species in the southwestern region. A phylogenetic tree was also constructed from the identified four species along with reference sequences of the blast results from the NCBI confirmed the four Solanaceae species i.e., Datura innoxia, Datura stramonium, Nicotiana glauca, and Withania somnifera, recorded invasive in the southern part of Saudi Arabia. Except for the matK tree, all the trees clustered individuals of the same species together and separately from the other species, with strong bootstrap support indicating that they were more closely related species.

Barcoding coffee grounds - Exploring pteropod gastropod biodiversity with dregs in collection jars

Despite their cosmopolitan occurrence and massive plankton sampling during expeditions, the genetic diversity within Pteropoda Cuvier, 1804 is still largely unexplored. In this study we present a next-generation environmental barcoding approach to zooplankton bulk samples, which were collected during the circumglobal 2010 Malaspina expedition to evaluate pteropod diversity. We introduce a technique that avoids destructive procedures and leaves material intact for further morphological investigations. We extracted DNA out of the dregs (organic material such as mucus or body parts) of 27 sample containers for molecular barcoding (average 100-260 bp of COI). We were able to identify 7128 operational taxonomic units corresponding to the species composition contained in the examined samples. Among them were three species of thecosome pteropods, Creseis acicula, Creseis virgula and Cavolinia inflexa, which are discussed with respect to their taxonomy and their geographic distribution. Unidentified gymnosomes were also present in our samples from warmer regions in oceanic waters of the southern Indian Ocean. To facilitate identification of species, it is beneficial to create a better database of pteropod COI barcodes. Furthermore, gathering environmental barcoding data on a broad global scale will help to better understand species abundance and distribution of pteropods in the world’s oceans, and potentially those of other planktonic organisms.

Marine biodiversity discovery: the metrics of new species descriptions

Based on the World Register of Marine Species (WoRMS), there are currently c. 242,000 known valid marine species living in the world's oceans and marine biota continue to be discovered and named steadily at a current average of 2,332 new species per year. The “average” newly described marine species is a benthic crustacean, annelid, or mollusc between 2 and 10 mm in size, living in the tropics at depths of 0–60 m, and represented in the description by 7–19 specimens. It is described after a shelf life of 13.5 years in an article with two to three authors in a journal with an IF <1, published by an academic institution or society or a small commercial publisher. It is highly likely that the description is not accompanied by molecular data and that its authors do not work in an institution in a region of the world where the new species comes from. At the current pace of discovery and characterization, it will take several hundred years to describe the remaining 1–2 million unknown marine species. With increased facilitation of access to literature, marine taxonomy will increasingly rely on retired professionals and citizen scientists. The barriers to new marine species descriptions are in part technological (access to habitats that are difficult to sample) and educational (training to generate and use molecular barcodes), but mostly institutional (funding of taxonomic work) and regulatory (restrictions imposed by access and benefit sharing legislation).

The microbial composition of larval airways from Drosophila melanogaster differ between specimens from laboratory and natural habitats

BackgroundThe fruit fly Drosophila melanogaster lives in natural habitats and has also long been used as a model organism in biological research. In this study, we used a molecular barcoding approach to analyse the airways microbiome of larvae of D. melanogaster, which were obtained from eggs of flies of the laboratory strain w1118 and from immune deficient flies (NF-kB-K), and from wild-caught flies. To assess intergenerational transmission of microbes, all eggs were incubated under the same semi-sterile conditions.ResultsThe airway microbiome of larvae from both lab-strains was dominated by the two families Acetobacteraceae and Lactobacillaceae, while larvae from wild-caught flies were dominated by Lactobacillaceae, Anaplasmataceae and Leuconostocaceae. Barcodes linked to Anaplasmataceae could be further assigned to Wolbachia sp., which is a widespread intracellular pathogen in arthropods. For Leuconostoceae, the most abundant reads were assigned to Weissella sp. Both Wolbachia and Weissella affect the development of the insects. Finally, a relative high abundance of Serratia sp. was found in larvae from immune deficient relish−/− compared to w1118 and wild-caught fly airways.ConclusionsOur results show for the first time that larvae from D. melanogaster harbor an airway microbiome, which is of low complexity and strongly influenced by the environmental conditions and to a lesser extent by the immune status. Furthermore, our data indicate an intergenerational transmission of the microbiome as shaped by the environment.

Morphological and Molecular Identification of Jujube Fruit Fly Carpomya incompleta (Becker, 1903) (Diptera: Tephritidae) in southeast Iraq

An accurate identification of an insect is essential for the effective pest management program. However, due to the restrictions of phenotypic identification, molecular process (e.g. DNA barcoding) is used to overcome some of these limitations. The present paper was aimed to identify the Jujube fruit fly by morphological characters of the adults and molecular DNA barcoding of the larval stage in southeast Iraq. The adults was morphologically identified as Carpomyia incompleta (Becker, 1903) (Diptera: Tephritidae). Results of the molecular method revealed that the mitochondrial COI – COII marker was successfully identified immature stage of the species. The sequence analysis results identified the specimens (AHL1 and AHL2) as C. incompleta. Sequences of the specimens were recorded in The US National Center for Biotechnology Information (NCBI) under accession numbers (ON045002.1 and ON045003.1, respectively. These results were consistent with the phenotypic identification of the Jujube fruit flies.

Molecular Barcoding: A Tool to Guarantee Correct Seafood Labelling and Quality and Preserve the Conservation of Endangered Species.

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.

New records and barcode sequence data of wood-inhabiting polypores in Benin with notes on their phylogenetic placements and distribution.

Wood-inhabiting fungi (WIF), such as polypores, are extremely species-rich and play vital roles in the functioning of forest ecosystems as decomposers. Despite the importance of polypores, our knowledge of the diversity and distribution of these fungi is still poor in general and especially for West Africa. To advance our knowledge we here summarise results from field collections between 2017 and 2021 and present (i) a taxonomic overview, (ii) phylogenetic placements and (iii) an illustrated catalogue of wood-inhabiting polypore fungi with colour pictures. During the field sampling campaigns, we collected 647 specimens. Based on morphological characteristics and molecular barcode data, 76 polypore species belonging to six orders, 15 families and 39 genera were identified. Of the 76 species, 30 are new to the West Africa, 69 new to Benin, and two new combinations Fuscoporia beninensis and Megasporia minuta are proposed. With this summary, we provide new data for further research. Citation: Olou BA, Langer E, Ryvarden L, Krah F-S, Hounwanou GB, Piepenbring M, Yorou NS (2023). New records and barcode sequence data of wood-inhabiting polypores in Benin with notes on their phylogenetic placements and distribution. Fungal Systematics and Evolution 11: 11-42. doi: 10.3114/fuse.2023.11.02.

METB-13. A SINGLE-CELL GENETIC IN VIVO LINEAGE-TRACING PLATFORM FOR MEDULLOBLASTOMA

Abstract Comparative single-cell studies of group 3/4 medulloblastomas (MBs) and fetal brain have identified a common hierarchy of glutamatergic-lineage cell types and an apparent cell-of-origin in the rhombic lip. An increased understanding of the genetic regulators of stem-cell maintenance and differentiation in MB would clearly be of therapeutic benefit. There is a significant need for lineage-tracing systems to model MB malignant transformation, progression, and response to therapy. We developed in vivo and in organoid genetic lineage-tracing systems with a single-cell readout. A high-complexity lentiviral library expressing heritable polyadenylated molecular barcodes was transduced into D283 and D425 patient-derived cell lines. Barcoded cells were injected into the brains of immunocompromised mice. Both preimplantation barcoded cultures and the resulting mature tumors were profiled by single-cell RNA-sequencing (scRNA-seq). This captured both endogenous RNA and barcode transcripts. While only minimal barcode clash was detected in the preimplantation cultures, we observed a clonal expansion of barcodes in mature tumors which aligned with phylogenetics analysis of expressed mutations in endogenous RNA. Methods for assessing lineage coupling between transcriptional clusters and inferring their lineage relationship were developed. Ongoing efforts to recover barcodes from tumor sections via spatial transcriptomics will be presented. These studies fill a gap in status quo models of MB which are needed to leverage recent findings derived from single-cell analysis of human tumors.

Molecular pathogen screening of louse flies (Diptera: Hippoboscidae) from domestic and wild ruminants in Austria

BackgroundHippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria.MethodsLouse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses.ResultsA total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida.ConclusionsMolecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.Graphical

An initial genomic blueprint of the healthy human oesophageal microbiome.

The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However, investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore explored the potential of culturomics and metagenomic binning to generate a catalogue of reference genomes from the healthy human oesophageal microbiome, alongside a comparison set from saliva. Twenty-two distinct colonial morphotypes from healthy oesophageal samples were genome-sequenced. These fell into twelve species clusters, eleven of which represented previously defined species. Two isolates belonged to a novel species, which we have named Rothia gullae. We performed metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in a recent study. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters, eight representing novel Candidatus species, which we have named Ca. Granulicatella gullae, Ca. Streptococcus gullae, Ca. Nanosynbacter quadramensis, Ca. Nanosynbacter gullae, Ca. Nanosynbacter colneyensis, Ca. Nanosynbacter norwichensis, Ca. Nanosynococcus oralis and Ca. Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria . Although members of the Patescibacteria are known to inhabit the oral cavity, this is the first report of their presence in the oesophagus. Eighteen of the metagenomic species were, until recently, identified only by hard-to-remember alphanumeric placeholder designations. Here we illustrate the utility of a set of recently published arbitrary Latinate species names in providing user-friendly taxonomic labels for microbiome analyses.Our non-redundant species catalogue contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples, 60 species occurred in at least one oesophageal metagenome from either study, with 50 identified in both cohorts. Recovery of genomes and discovery of new species represents an important step forward in our understanding of the oesophageal microbiome. The genes and genomes that we have released into the public domain will provide a base line for future comparative, mechanistic and intervention studies.

Performance of SNP barcodes to determine genetic diversity and population structure of Plasmodium falciparum in Africa.

Panels of informative biallelic single nucleotide polymorphisms (SNPs) have been proposed to be an economical method to fast-track the population genetic analysis of Plasmodium falciparum in malaria-endemic areas. Whilst used successfully in low-transmission areas where infections are monoclonal and highly related, we present the first study to evaluate the performance of these 24- and 96-SNP molecular barcodes in African countries, characterised by moderate-to-high transmission, where multiclonal infections are prevalent. For SNP barcodes it is generally recommended that the SNPs chosen i) are biallelic, ii) have a minor allele frequency greater than 0.10, and iii) are independently segregating, to minimise bias in the analysis of genetic diversity and population structure. Further, to be standardised and used in many population genetic studies, these barcodes should maintain characteristics i) to iii) across various iv) geographies and v) time points. Using haplotypes generated from the MalariaGEN P. falciparum Community Project version six database, we investigated the ability of these two barcodes to fulfil these criteria in moderate-to-high transmission African populations in 25 sites across 10 countries. Predominantly clinical infections were analysed, with 52.3% found to be multiclonal, generating high proportions of mixed-allele calls (MACs) per isolate thereby impeding haplotype construction. Of the 24- and 96-SNPs, loci were removed if they were not biallelic and had low minor allele frequencies in all study populations, resulting in 20- and 75-SNP barcodes respectively for downstream population genetics analysis. Both SNP barcodes had low expected heterozygosity estimates in these African settings and consequently biased analyses of similarity. Both minor and major allele frequencies were temporally unstable. These SNP barcodes were also shown to identify weak genetic differentiation across large geographic distances based on Mantel Test and DAPC. These results demonstrate that these SNP barcodes are vulnerable to ascertainment bias and as such cannot be used as a standardised approach for malaria surveillance in moderate-to-high transmission areas in Africa, where the greatest genomic diversity of P. falciparum exists at local, regional and country levels.

Liquid biopsy in combination with PET/CT versus PET/CT alone in diagnosis of lung cancer in small nodules.

3072 Background: In the National Lung Screening Trial, almost two-thirds of patients that underwent an invasive procedure for diagnosis had benign findings, suggesting a significant number of patients were exposed to risk that could be avoided. Currently, to help distinguish malignant from benign nodules, Positron Emission Tomography/Computed Tomography (PET/CT) is used. However, PET/CT can be limited in its use for lesions less than 2 cm in size. The detection of circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) in the blood stream via liquid biopsy has demonstrated potential as a non-invasive approach in both screening and diagnostic tests. The purpose of this study is to assess whether combining liquid biopsy with PET/CT findings can better detect malignancy in small lung nodules 6-20 mm in size compared to PET/CT alone, possibly allowing earlier cancer detection and reduction in unneeded invasive diagnostic testing. Methods: This was a single-center prospective observational study for which the primary objective was to compare liquid biopsy plus PET/CT to PET/CT alone for detection of lung cancer in patients with newly discovered or newly growing solid lung nodules. Patients with nodules 6-20 mm in size who were scheduled to undergo 18F-Fluorodeoxyglucose PET/CT also underwent blood collection for plasma ctDNA/cfDNA testing using an amplicon-based next-generation sequencing assay incorporating molecular barcodes for error suppression. Tumor diagnosis was based on resolution of the nodule or tissue biopsy. For liquid biopsy, a multiomic model previously built using (1) cfDNA methylation frequencies, (2) ctDNA abundance, (3) somatic ctDNA mutations, and (4) cfDNA fragment size ratio was used to predict malignancy. For imaging, lesions with a maximum standardized uptake value of 2 or greater were considered malignant. Results: Between December 2021 and November 2022, 24 patients were enrolled. Of these, 13 had a definitive diagnosis and were included in the analysis with the remainder still undergoing serial imaging follow-up. Of the 13 patients, 9 were biopsy or surgically proven to be malignant, and 4 were benign based on biopsy or resolution. The median (IQR) nodule size was 14 mm (8-15). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for PET/CT were 0.67, 1, 1, and 0.57, respectively. For multiomic liquid biopsy alone, these values were the same. However, combining multiomic liquid biopsy and PET/CT, the sensitivity, specificity, PPV, and NPV were 0.89, 1, 1, and 0.8, respectively. Conclusions: Combining multiomic liquid biopsy with PET/CT results in improved sensitivity for the detection of early-stage malignant solid lung nodules by 33% compared to PET/CT alone with no reduction in specificity. This could result in improved selection of patients requiring biopsy. Further evaluation with a larger cohort is needed to confirm the findings.

First report of ELUCIDATOR study: A prospective observational multicenter study on resistance mechanisms in patients with early disease progression during osimertinib.

3032 Background: Osimertinib (Osi) is the key standard therapy for patients (pts) with chemo-naive advanced non-small cell lung cancer (NSCLC) harboring sensitizing epidermal growth factor receptor ( EGFR) mutations. However, many of cases treated with Osi experience the disease progression (PD). Therefore, understanding first-line osimertinib resistance mechanisms is essential for future therapeutic strategies. Here, we addressed the underlying mechanisms of resistance to Osi using paired plasma DNA samples and particularly focused on early PD (PD within 6 months) cases. Methods: This trial is the multicenter prospective research, and we prospectively collected plasma DNA samples prior Osi treatment and at PD. Next-generation sequencing (NGS) implemented with the molecular barcoding method was performed to analyze gene mutations in 197 cancer-associated genes. Then using these ct-DNA, we analyzed the Osi resistance by ultra-sensitive NGS. In this interim analysis, we specially analyze the early PD (PD within 6 months) cases. Results: One hundred eighty-eight pts were enrolled between May 2019 and January 2021. Among these pts, 125 (66%) pts were female, 96 (51%) were EGFR exon 19 deletion mutation, and 109 (58%) were never smoker. Seventy-eight pts experienced PD and 36 pts experienced early PD at interim analysis. Baseline shedders, patients with plasma positivity for EGFR-activating mutation were more frequently observed in early PD pts than non-PD (86.1% vs 63.4%, p = 0.009). Early PD patients had significantly more co-occurring gene mutations along with EGFR mutations than non-early PD patients (2.89±1.49 vs 1.97±1.37, p = 0.002). Most notably, 3 among 36 early PD pts and 8 among 78 PD pts had significant reduction of plasma EGFR mutant allele frequency at PD than that at baseline. Among those, 1 had germline BRCA1 mutation and 1 had BRINP3 mutation in 3 early PD pts, and 1 had germline BRCA2 mutation, and 1 had germline TP53 mutation in 5 PD pts excluding early PD pts. Co-occurring TP53 mutations were not relevant to early PD in this study cohort. Conclusions: Germline mutations in BRCA1/2, TP53 and BRINP3 may confer early and unprecedented resistance to Osi monotherapy. Plasma-based serial comprehensive gene profiling may help predict and identify patients unlikely to benefit from Osi treatment, though further study is warranted. Clinical trial information: jRCTs031180051 .

High diversity of aquatic Sparganium (Xanthosparganium, Typhaceae) in North Eurasia is mostly explained by recurrent hybridization

The species composition and extent of hybridization in Sparganium subgenus Xanthosparganium in North Eurasia reported in different published sources significantly vary. Thus, we aimed to clarify the taxonomy and distribution of aquatic Sparganium in that area. We supplemented the existing fragmentary genetic and morphological data mainly from North America and South Asia with our data from East Europe and North Asia. We combined molecular barcoding of the nuclear phyC and plastid psbJ-petA DNA regions (382 samples) with morphological analysis of herbarium collections (more than 1500 specimens from 16 herbaria) and numerous natural populations with a special focus on hardly accessible Siberian and the Far Eastern regions of Russia. We found that aquatic Sparganium is represented in North Eurasia by nine species and 14 hybrids. Nine previously unknown hybrids are formally described as new nothotaxa. All species and hybrids could be reliably discriminated with barcoding. We refined the distribution of all taxa in North Eurasia, e.g., S. angustifolium, a species avoiding continental areas, where it was confused by many authors with mostly vegetative specimens of other taxa. In the S. emersum complex in addition to recognized earlier widespread S. emersum and eastern North American S. chlorocarpum we proved the existence of one more distinct lineage – Asian Pacific S. rothertii. We discovered different evolutionary lineages within some species (e.g., S. glomeratum and S. hyperboreum) causing additional issues in the taxa identification. Almost all species cross with each other, usually acting both as plastid and pollen donors. Most of the hybrids are widespread and abundant. They originate each time when the ranges of parental species overlap and suitable habitats are available, and rather do not disperse from the centres of origin. Hybridization can be a threat to species with narrow ecological tolerance. Active gene flow is also evident within species when different evolutionary lineages come in contact (e.g., S. emersum, S. rothertii, S. glomeratum, S. hyperboreum, S. natans). We provide a new taxonomic treatment, which solves many long-standing issues in subgenus Xanthosparganium, and a new identification key for both species and hybrids occurring in North Eurasia.