DNA Analysis Research Articles

Complete mitochondrial genomes and phylogenetic analysis of native and non-native fishes in a national key wetland of China

Fish are considered objective indicators of environmental health and ecosystem stability. Establishing regional reference databases of mitochondrial genome sequences from local fish communities can significantly enhance fish monitoring using environmental DNA (eDNA) analysis. For non-native species, the eDNA technique provides early detection and rapid monitoring. It is also crucial to include fundamental genetic information for both native and non-native species in genetic databases. This study presents the complete mitochondrial genomes of 17 fish species inhabiting the Baiyangdian Basin, a national key wetland in China. The mitochondrial DNA of these fish was analyzed to investigate their characteristics, and their phylogeny was determined using maximum likelihood (ML) methods. Various analyses were performed, including the examination of nucleotide composition, evaluation of AT-skew and GC-skew, analysis of codon frequency, and determination of relative synonymous codon usage (RSCU) values, and assessment of selection pressure on protein-coding genes (PCGs). The analysis showed that all PCGs in all fish underwent purifying selection. Using Xenocyprididae as a representative, this study investigated the genetic selection tendencies of native and non-native fish species in the Baiyangdian Basin. Significant differences were found in five of the 13 PCGs: COI, COII, COIII, Cytb, and ATP8. Except for ATP8, the findings indicated that the genes of non-native species underwent stronger purifying selection during evolution compared to native species. Additionally, comparing the population ω values of non-native species to those of native species showed that the Cytb and COIII genes exhibited greater differential purification selection than COI and COII. These differences may be the result of the evolution of non-native species to migrate and adapt to the Baiyangdian Basin, thereby affecting the evolution of related genes.

"This Time It Was Different:" Creating a Multidisciplinary, Trauma-Informed, Victim-Centered Approach to Sexual Assault Cold Case Investigations and Prosecutions.

Police and prosecutors recommend that sexual assault survivors have a medical forensic exam and the collection of a sexual assault kit (SAK; also known as a "rape kit") to preserve biological evidence (e.g., semen, blood, saliva, hair) if they want to pursue criminal prosecution. However, law enforcement personnel do not routinely submit SAKs to crime laboratories for forensic DNA testing. Instead, they often place untested kits in storage and close many of these reported cases after minimal investigation. Current estimates indicate there are 300,000 to 400,000 untested SAKs in law enforcement agencies throughout the United States. In response to this national problem, the U.S. Department of Justice created the Sexual Assault Kit Initiative (SAKI) Project to support kit testing, re-investigation, and prosecution of these "cold case" sexual assaults. The SAKI program also provides training and technical assistance to police, prosecutors, and victim advocates on how to use a multidisciplinary, trauma-informed, and victim-centered approach in cold case prosecutions. This study examined the extent to which one SAKI-funded site implemented these three guiding principles in their interactions with victims while prosecuting cold case sexual assaults. We conducted semistructured qualitative interviews with N = 32 sexual assault survivors from the first cohort of cold cases that were re-opened and prosecuted in this jurisdiction. Nearly all cases (n = 31) ended in a guilty plea or trial conviction, and the vast majority of survivors indicated that they had positive experiences with the SAKI team. Survivors noted that they were listened to, believed, supported, and well-prepared by a multidisciplinary team of practitioners who were personally invested in their cases and in their well-being. Implications for creating multidisciplinary, trauma-informed, and victim-centered approaches with other communities are discussed.

Assessment of the genetic polymorphism of genus Pinus L. the oligotrophic peat bog "Boloto Mshana"

Aim. To evaluate the molecular genetic polymorphism of representatives of the genus Pinus L. on the territory of the "Boloto Mshana" nature reserve, as well as to test the hypothesis about their possible local hybridization Methods. DNA extraction using CTAB and DNeasy Plant Pro Kit, polymerase chain reaction (PCR) with primers for the chloroplast DNA and genes encoding β-tubulin (TBP markers), electrophoresis of DNA in a polyacrylamide gel with silver nitrate staining. Results. DNA profiles of P. mugo, P. sylvestris and probably P. uliginosa (21 samples in total) were analyzed using one chloroplast DNA marker and two DNA markers based on introns of β-tubulin genes (ТВР, сТВР). The intraspecific genetic polymorphism of the species was compared and the probability of the existence of hybrid forms was investigated. Conclusions. The marker based on the 2nd intron of the β-tubulin genes turned out to be the most effective and informative DNA marker for studying representatives of the Pinus L. genus. P. sylvestris showed less genetic polymorphism by the investigated genetic markers, compared to P. mugo and P. uliginosa. Possible hybrid forms (P. mugo ✕ P. sylvestris) or P. uliginosa were also discovered.

FamLink2 – A comprehensive tool for likelihood computations in pedigrees analyses involving linked DNA markers accounting for genotype uncertainties

There is an increasing demand for software that can handle an arbitrary number of linked markers in forensic genetics; primarily with application to inference of relationships and direct matching but also in applications such as ancestry inference and mixture interpretation. With the emergence of sequencing technologies, denser sets of SNP markers are generated and analyzed. Additionally, sequence data of low quality and quantity DNA generate uncertainty about the underlying true genotype. We provide an efficient implementation of a general model for pedigree likelihood computations with genetic marker data using a three-layered approach. The top and first layer is the population model where allele frequencies and population substructure are accounted for. The second layer is the inheritance model which efficiently handles linked markers using an IBD model. The third and bottom layer is the observational level where we model the likelihood of the true genotype given underlying reads as well as parameters for errors. We exemplify the utility of our implementation as well as provide validation according to guidelines established by the ISFG using a combination of two published SNP panels. We demonstrate that computations are feasible for panels encompassing 10,000 markers and we argue that, due to the properties of the underlying algorithm, extending the number of markers will result in a linear increase in computation time. In addition we study the impact of parameters used in our model and suggest some guidelines pertaining to their values. The results demonstrate that a probabilistic model for low coverage sequence read data is needed instead of relying on an a threshold based genotype and applying our general model for inference of relationships on a real case can be superior, i.e. higher information content, to other methods relying on either fixed genotypes with low quality sequence data or simple pair wise relationship tests. In summary, the implementation, FamLink2 (freely available at https://famlink.se), can jointly handle genetic linkage, genotype uncertainty and population substructure for an arbitrary pedigree with data for any number of individuals. Whereas the current study will focus on calculations disregarding mutations, FamLink2 has the ability to model mutations for certain built-in pedigrees.

Aspects of NCBI GenBank as a Biodiversity Information Resource

DNA sequencing of museum specimens, also known as museomics, provides new insights into the study of biodiversity, including taxonomy, phylogeny, and environmental studies. Also, sequencing specimens have led to the rediscovery of extinct species (Suzuki et al. 2016), identification of related species (Waku et al. 2016), and analysis of ancient DNA (Kanzawa-Kiriyama et al. 2016). Nucleotide sequence data have been collected for more than 30 years under the framework of the International Nucleotide Sequence Database Collaboration (INSDC) by three institutes, namely, National Center for Biotechnology Information, US (NCBI), European Bioinformatics Institute (EBI), and DNA Data Bank of Japan (DDBJ) (Arita et al. 2020). NCBI has collated a database of sequence data, GenBank, which contains approximately 494 million sequences as of April 2022 (Sayers et al. 2021). In fact, GenBank is designed with qualifiers to describe various types of biodiversity information such as "/specimen_voucher", "/lat_lon" (latitude and longitude) and "/collection_date". Also, INSDC now requires that all submissions include the sampling location and date (INSDC 2023). I surveyed the biodiversity information assigned to GenBank records to determine the potential of GenBank as a biodiversity resource. I downloaded all GenBank data as of August 2023 from the FTP site. The “/specimen_voucher” qualifier was introduced to describe specimen ID in Release 104 in December 1997. This qualifier was designed to fill the value in free text: for example, /specimen_voucher="Smith s. n. 4-IV-1995 (U. S. Natl. Herbarium)". After Release 162 in October 2007, a method of writing with a structured value of "[<institution-code>: [<collection-code>:]] <specimen_id>" was added (institution-code and collection-code are optional). There are 527,215 records (37.8%) with "/specimen_voucher" qualifier for fish, 3,096,112 records (40.3%) for insects, 1,505,556 records (39.0%) for flowering plants. But fewer than 10% of records have specimen IDs listed using this structured description. To utilize these ambiguous specimen IDs in GenBank, these IDs may need to be cleansed using databases such as NCBI BioCollections, GRSciColl (Global Registry of Scientific Collections) or AI to map them to IDs in databases rich in specimen information such as those of the Global Biodiveristy Information Facility (GBIF) and Barcode of Life System (BOLD). In GenBank, the BOLD ID is listed in the /db_xref qualifier in the “Features” field as the ID of the external database. The 70% of insect sequence data with a specimen ID in the /specimen_voucher qualifier are also assigned a BOLD ID (Nakazato and Jinbo 2022). The correspondence between specimen IDs in biodiversity information databases such as GBIF and specimen IDs in GenBank is expected to further enhance the value of museum specimens. In addition, GenBank provides the /type_material qualifier for describing the type of voucher (e.g., holotype of Asphondylia bursicola). In GenBank insect data, there were over 2,000 records for type material, and approximately 450 species were mentioned, including 269 for holotypes. We found approximately 3,000 records with type information by including “/notes” and “/specimens_voucher” qualifiers in addition to “/type_material”. Thus, GenBank has potential as a biodiversity information resource, but for more effective use, data mining and linkage with other specimen-based biodiversity databases are essential.

Mitochondrial DNA analysis reveals urgent conservation needs for the southernmost population of ayu (Plecoglossus altivelis)

Mitochondrial DNA analysis reveals urgent conservation needs for the southernmost population of ayu (Plecoglossus altivelis)

Evaluating the quantitative performance of environmental DNA metabarcoding for freshwater zooplankton community: a case study in Lake Biwa, Japan.

Zooplankton monitoring is important for understanding their population dynamics and life history, ecosystem health, and environmental changes. Compared with traditional morphological identification, environmental DNA (eDNA) analysis allows for more sensitive and efficient monitoring of zooplankton diversity. Previous eDNA studies have primarily used metabarcoding approaches to reveal their richness and composition, whereas its performance in predicting zooplankton abundance remains understudied. We conducted water and bulk sampling in Lake Biwa, Japan, showing that the number of sequence reads by metabarcoding moderately correlated with eDNA concentrations estimated by quantitative real-time PCR (qPCR). In addition, the eDNA read number was significantly related to cladoceran and copepod abundance estimated by microscopy sorting, although there remained too much uncertainty in the read-abundance relationship. Moreover, there was a significant difference in species composition between eDNA metabarcoding and sorting. Although our results indicated the potential applicability of eDNA metabarcoding for quantifying multiple zooplankton abundance, several methodological validations in eDNA metabarcoding would also be required to optimize its performance in the future.

Fluorescent sensing platform based on two-dimensional layered double hydroxides and exonuclease I for DNA and microRNA detection

Interfacial and interlayer interactions between layered double hydroxides (LDHs) and nucleic acids offer LDHs great potential to absorb and detect nucleic acids. Herein, we demonstrate a simple, rapid, and versatile fluorescent sensing platform based on two-dimensional LDHs and exonuclease I (Exo I) for the selective analysis of DNA and microRNA. ZnNiAl-LDHs were utilized as quenchers for FAM labelled on single-stranded DNA (ssDNA) probes via dynamic quenching with high fluorescence quenching ability. The quenching mechanism was explored by means of spectral analysis, characterization experiments, DNA adsorption and desorption studies, zeta potential analysis, and molecular dynamics simulations. The driving forces for the adsorption of LDHs toward fluorophore-labelled nucleic acids were confirmed to be electrostatic interaction, van der Waals force, and anion exchange for the first time. In order to distinguish the formed target/probe duplexes from FAM-ssDNA probes, Exo I was introduced to specifically hydrolyze FAM-ssDNA probes. The fabricated fluorescent sensing platform for nucleic acids detection shows a low detection limit of 0.08 nM. Finally, this sensor was effectively utilized for the detection of DNA and microRNA in actual biological and environmental samples, achieving recoveries ranging from 96.4 % to 105 %, indicating high accuracy. This work proposed a promising sensing platform for sequence-specific nucleic acids detection, and provides a new perspective on two-dimensional LDHs as structural supports and biomolecular reservoirs for biomedical applications.

The effect of insect excretions/secretions and decomposition fluid on DNA quantity and quality in human bloodstains.

The larval excretions/secretions (ES) of blowflies contain proteolytic enzymes and bacteria that assist with tissue breakdown. Decomposition fluid (DF) contains organic and inorganic waste products from cell death. This study investigated if human DNA recovery from blood was impacted by exposure to ES and DF over time. Lucilia sericata ES were collected daily from 50 larvae, and all available DF was collected from two fetal piglets left to decompose for 2 weeks. Daily for 3-5 days, 28 μL-30 μL of ES, DF, or a 1:1 mixture of the fluids was added to 30 μL of blood on cotton. Three bloodstains per treatment were sampled every 12 h up to 3 days and at 1 and 2 weeks after initial addition of fluid. No PCR inhibition was detected, but DNA degradation increased over time, primarily in samples exposed to ES and ES/DF mixtures. The amount of DNA recovered decreased over time, but generally more DNA was recovered from DF samples than other samples. Full profiles, or partial profiles suitable for routine database searching (14-39 alleles), were generated from all DF and ES samples and at least one mixture sample at all timepoints. Partial profiles of between 1 and 13 alleles were obtained from all other mixture samples, except one mixture sample which generated no profile. These findings indicate bloodstain evidence recovered from maggot-infested and/or decomposing bodies may generate forensically useful DNA evidence and should be analyzed as quickly as possible after collection or stored appropriately to prevent further degradation.

TigerBase: A DNA registration system to enhance enforcement and compliance testing of captive tiger facilities

The illegal trade in tigers (Panthera tigris) and their derivatives, such as bones, teeth and pelts, is a major threat to the species’ long-term persistence. As wild tiger populations have dwindled, a large proportion of trafficked tiger products now derive from captive breeding facilities found throughout Asia. Moreover, wild tigers have been poached and laundered into captive facilities, then falsely designated as captive-bred. The establishment of a DNA registration system is recognized as a key tool to monitor compliance of captive facilities, support tiger trade investigations and improve prosecution outcomes. Here, we present a standardised wildlife forensic DNA profiling system for captive tigers called TigerBase. TigerBase has been developed in four South-East Asia countries with captive tiger facilities: Malaysia, Vietnam, Thailand and Lao PDR. TigerBase DNA profile data is based on 60 single nucleotide polymorphism (SNP) markers, genotyped using two different TaqMan®-based approaches: OpenArray® chip (capable of genotyping 60 SNPs for 48 samples in a single chip), and singleplex TaqMan® assays (capable of genotyping one SNP for one sample per reaction). Of the 60 SNPs, 53 are autosomal nuclear markers, suitable for individualisation and parentage applications, two are sex-linked markers, suitable for sexing, and five are mtDNA markers, suitable for maternal subspecies identification. We conducted a series of validation experiments to investigate the reliability and limitations of these SNP genotyping platforms. We found that the OpenArray® chip platform is more appropriate for generating reference data given its greater throughput, while the singleplex TaqMan® assays are more appropriate for genotyping lower quality casework samples, given their higher sensitivity and throughput flexibility. Only 19 autosomal nuclear markers were validated as singleplex TaqMan® assays, which generally provides ample power for individualisation analysis (probability of identity among siblings was <6.9 ×10−4), but may lack power for specific parentage questions, such as determining parentage of an offspring when one of the parent’s genotypes is missing. Further, we have developed pipelines to support standardised SNP calling and decrease the chance of genotyping errors through the use of analytical workflows and synthetic positive controls. We expect the implementation of TigerBase will enhance enforcement of tiger trafficking cases and encourage compliance among captive tiger facilities, together contributing to combatting the illegal tiger trade.

Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn7016 transposon (PseCAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. PseCAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.

Prenatal diagnostic errors in hemoglobin Bart's hydrops fetalis caused by rare genetic interactions of α-thalassemia.

To describe rare genetic interactions of α-thalassemia alleles causing Hb H disease and Hb Bart's hydrops fetalis which could lead to diagnostic errors in a routine practice. Hematological and molecular characterization were carried out in a Thai family with a risk of having fetus with Hb Bart's hydrops fetalis. Both parents were found to be the thalassemia intermedia patients associated with unusual forms of Hb H disease. DNA analysis of common α-thalassemia mutations in Thailand identified α+-thalassemia (-α3.7 kb del) and unknown α0-thalassemia in the father and α0-thalassemia (--SEA) with unknown α+-thalassemia in the mother. Fetal DNA analysis unlikely identified a homozygosity for α0-thalassemia (--SEA/--SEA). Further analysis identified that the father carried a rare South African α0-thalassemia in combination with α+-thalassemia (--SA/-α), whereas the mother was a patient with Hb H-Queens Park disease (--SEA/ααQP). The fetus was, in fact, a compound heterozygote for (--SA/--SEA). As shown in this study, routine screening for α-thalassemia at prenatal diagnosis in the region should include both common and rare α0-thalassemia alleles found in the population to effectively prevent a fatal condition of Hb Bart's hydrops fetalis syndrome.

Transcriptional profiling in microglia across physiological and pathological states identifies a transcriptional module associated with neurodegeneration

Microglia are the resident immune cells of the central nervous system and are involved in brain development, homeostasis, and disease. New imaging and genomics technologies are revealing microglial complexity across developmental and functional states, brain regions, and diseases. We curated a set of publicly available gene expression datasets from human microglia spanning disease and health to identify sets of genes reflecting physiological and pathological microglial states. We also integrated multiple human microglial single-cell RNA-seq datasets in Alzheimer’s disease (AD), multiple sclerosis (MS), and Parkinson’s disease, and identified a distinct microglial transcriptional signature shared across diseases. Analysis of germ-line DNA identified genes with variants associated with AD and MS that are overrepresented in microglial gene sets, including the disease-associated transcriptional signature. This work points to genes that are dysregulated in disease states and provides a resource for the analysis of diseases in which microglia are implicated by genetic evidence.

Bacteriophage λ exonuclease and a 5'-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids.

Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ imaging. Clustered regularly interspaced short palindromic repeats and Cas systems rely on protospacer-adjacent motif (PAM)-dependent double-stranded DNA (dsDNA) recognition, limiting the range of targetable sequences and leading to undesired off-target effects. Using single-molecule fluorescence resonance energy transfer analysis, we discover the enzymatic activity of bacteriophage λ exonuclease (λExo). We show binding of 5'-phosphorylated single-stranded DNA (pDNA) to complementary regions on dsDNA and DNA-RNA duplexes, without the need for a PAM-like motif. Upon binding, the λExo-pDNA system catalytically digests the pDNA into nucleotides in the presence of Mg2+. This process is sensitive to mismatches within a wide range of the pDNA-binding region, resulting in exceptional sequence specificity and reduced off-target effects in various applications. The absence of a requirement for a specific motif such as a PAM sequence greatly broadens the range of targets. We demonstrate that the λExo-pDNA system is a versatile tool for molecular diagnostics, DNA computing and gene imaging applications.

Characterization of four novel actinospore types of fish parasitic myxozoans and the occurrence of Branchiodrilus hortensis and Ophidonais serpentina from fish farms of Hungary

Six distinct actinospore types were identified in the intestinal epithelium of oligochaetes collected from the Szigetbecse and Makád fish farms of Ráckeve Danube Arm Fishing Association, in Hungary. Four new types: triactinomyxon type, raabeia type, aurantiactinomyxon type 1, and aurantiactinomyxon type 2, were described morphologically and molecularly from three invertebrate host species: Limnodrilus hoffmeisteri, Ophidonais serpentina, and Tubifex tubifex. The 18S ribosomal DNA (rDNA) analysis revealed that these new types of actinospores did not match any myxospore sequences available in GenBank. Phylogenetic analysis placed the triactinomyxon type within the Myxobolus clade that parasitizes cyprinid fish, while the raabeia type and aurantiactinomyxon type 2 were both placed within the Myxobolus clade associated with Perciformes fish. Aurantiactinomyxon type 1 was clustered in a clade containing gill-infecting Henneguya spp. from Esociformes fish. However, no myxospores have been found yet to link to the newly sequenced actinospores reported in this survey. This study also reports the first occurrence of two oligochaetes species, Branchiodrilus hortensis and Ophidonais serpentina in Hungary, specifically in fish farms of Ráckeve Danube Arm Fishing Association. Moreover, this is the first report on the involvement of Ophidonais serpentina in the life cycle of myxozoans.

Long-read de novo genome assembly of Gulf toadfish (Opsanus beta)

BackgroundThe family Batrachoididae are a group of ecologically important teleost fishes with unique life histories, behavior, and physiology that has made them popular model organisms. Batrachoididae remain understudied in the realm of genomics, with only four reference genome assemblies available for the family, with three being highly fragmented and not up to current assembly standards. Among these is the Gulf toadfish, Opsanus beta, a model organism for serotonin physiology which has recently been bred in captivity.ResultsHere we present a new, de novo genome and transcriptome assemblies for the Gulf toadfish using PacBio long read technology. The genome size of the final assembly is 2.1 gigabases, which is among the largest teleost genomes. This new assembly improves significantly upon the currently available reference for Opsanus beta with a final scaffold count of 62, of which 23 are chromosome scale, an N50 of 98,402,768, and a BUSCO completeness score of 97.3%. Annotation with ab initio and transcriptome-based methods generated 41,076 gene models. The genome is highly repetitive, with ~ 70% of the genome composed of simple repeats and transposable elements. Satellite DNA analysis identified potential telomeric and centromeric regions.ConclusionsThis improved assembly represents a valuable resource for future research using this important model organism and to teleost genomics more broadly.

Biosynthetic gene clusters from uncultivated soil bacteria of the Atacama Desert.

Soil microorganisms mediate several biological processes through the secretion of natural products synthesized in specialized metabolic pathways, yet functional characterization in ecological contexts remains challenging. Using culture-independent metagenomic analyses of microbial DNA derived directly from soil samples, we examined the potential of biosynthetic gene clusters (BGCs) from six bacterial communities distributed along an altitudinal gradient of the Andes Mountains in the Atacama Desert. We mined 38 metagenome-assembled genomes (MAGs) and identified 168 BGCs. Results indicated that most predicted BGCs were classified as non-ribosomal-peptides (NRP), post-translational modified peptides (RiPP), and terpenes, which were mainly identified in genomes of species from Acidobacteriota and Proteobacteria phyla. Based on BGC composition according to types of core biosynthetic genes, six clusters of MAGs were observed, three of them with predominance for a single phylum, of which two also showed specificity to a single sampling site. Comparative analyses of accessory genes in BGCs showed associations between membrane transporters and other protein domains involved in specialized metabolism with classes of biosynthetic cores, such as resistance-nodulation-cell division (RND) multidrug efflux pumps with RiPPs and the iron-dependent transporter TonB with terpenes. Our findings increase knowledge regarding the biosynthetic potential of uncultured bacteria inhabiting pristine locations from one of the oldest and driest nonpolar deserts on Earth.IMPORTANCEMuch of what we know about specialized metabolites in the Atacama Desert, including Andean ecosystems, comes from isolated microorganisms intended for drug development and natural product discovery. To complement research on the metabolic potential of microbes in extreme environments, comparative analyses on functional annotations of biosynthetic gene clusters (BGCs) from uncultivated bacterial genomes were carried out. Results indicated that in general, BGCs encode for structurally unique metabolites and that metagenome-assembled genomes did not show an obvious relationship between the composition of their core biosynthetic potential and taxonomy or geographic distribution. Nevertheless, some members of Acidobacteriota showed a phylogenetic relationship with specific metabolic traits and a few members of Proteobacteria and Desulfobacterota exhibited niche adaptations. Our results emphasize that studying specialized metabolism in environmental samples may significantly contribute to the elucidation of structures, activities, and ecological roles of microbial molecules.

Halorubrum miltondacostae sp. nov., a potential polyhydroxyalkanoate producer isolated from an inland solar saltern in Rio Maior, Portugal

One hundred and sixty-three extreme halophiles were recovered from a single sample collected from an inland solar saltern in Rio Maior. Based on random amplified polymorphic DNA (RAPD) profiles and partial 16S rRNA gene sequencing 125 isolates were identified as members of the Archaea domain within the genus Halorubrum. Two strains, RMP-11T and RMP-47, showed 99.1 % sequence similarity with the species Halorubrum californiense based on phylogenetic analysis of the 16S rRNA gene sequence. However, phylogenetic analysis based on five housekeeping genes, atpB, EF-2, glnA, ppsA and rpoB’, showed Halorubrum coriense as the closest related species with 96.7 % similarity. The average nucleotide identity (ANI) of strains RMP-11T, RMP-47 and species Hrr. coriense were within the range of 90.0–90.5 %, supporting that strains RMP-11T and RMP-47 represent a novel species of the genus Halorubrum. These strains formed red-pigmented colonies that were able to grow in a temperature range of 25–50 °C. Polyhydroxyalkanoate (PHA) granules were detected in both strains. The polar lipid profile was identical to the neutrophilic species of the genus Halorubrum. The Rio Maior sample from which both strains were isolated was metagenome sequenced. We identified five metagenome-assembled genomes representing novel Halorubrum species but distinct from the species represented by strains RMP-11T and RMP-47. Based on phylogenetic, phylogenomic, comparative genomics, physiological and chemotaxonomic parameters, we describe a new species of the genus Halorubrum represented by strains RMP-11T (=CECT 30760T = DSM 115521T) and RMP-47 (=CECT 30761 = DSM 115541) for which we propose the name Halorubrum miltondacostae sp. nov.

The Barren Berry phenotype: untangling cranberry (Vaccinium macrocarpon Ait.) genetic contamination in central Wisconsin and beyond

Wisconsin is the world’s leading producer of cranberries (Vaccinium macrocarpon Ait.; 2n = 2x = 24). The state produces over twenty-thousand acres that contribute to more than 50% of the global total production, with more than one billion dollars in value. Cranberry growers in the “central sands” of Wisconsin have been experiencing yield decline due to vegetative unproductive genotypes, popularly known as “Barren Berry’’, which consistently remain vegetative and produce no fruit. The purpose of this study was to compare visual inspection in the field to DNA fingerprinting for the early detection of unproductive/barren genotypes. Additionally, the study served as a survey of unproductive genotypes in central Wisconsin, the largest growing region in the world. A total of 839 cranberry leaf samples, from 14 growers representing plantings to four cultivated varieties, were submitted for DNA testing of two visually determined phenotypes: unproductive or barren (n = 646; those that produced little to no fruit), versus productive or fruiting (n = 193). We conducted genetic fingerprinting on the leaf tissue using nine microsatellite markers previously shown to differentiate cranberry genotypes. This study identified a barren berry genotype unique to central Wisconsin, which we denoted “Barren Berry 1”. This genotype accounted for 43% of samples submitted as the barren berry phenotype. Genetic fingerprinting revealed sixty-five different genotypes in beds which were thought to be monocultures of just four cultivated genotypes. Early detection of genetic contamination through visual inspection, genetic testing, and responsible propagation can drastically aid in the management and longevity of cranberry beds, and in turn save time and money to the growers.

Targeted therapy guided by circulating tumor DNA analysis in advanced gastrointestinal tumors.

Although comprehensive genomic profiling has become standard in oncology for advanced solid tumors, the full potential of circulating tumor DNA (ctDNA)-based profiling in capturing tumor heterogeneity and guiding therapy selection remains underexploited, marked by a scarcity of evidence on its clinical impact and the assessment of intratumoral heterogeneity. The GOZILA study, a nationwide, prospective observational ctDNA profiling study, previously demonstrated higher clinical trial enrollment rates using liquid biopsy compared with tissue screening. This updated analysis of 4,037 patients further delineates the clinical utility of ctDNA profiling in advanced solid tumors, showcasing a significant enhancement in patient outcomes with a 24% match rate for targeted therapy. Patients treated with matched targeted therapy based on ctDNA profiling demonstrated significantly improved overall survival compared with those receiving unmatched therapy (hazard ratio, 0.54). Notably, biomarker clonality and adjusted plasma copy number were identified as predictors of therapeutic efficacy, reinforcing the value of ctDNA in reflecting tumor heterogeneity for precise treatment decisions. These new insights into the relationship between ctDNA characteristics and treatment outcomes advance our understanding beyond the initial enrollment benefits. Our findings advocate for the broader adoption of ctDNA-guided treatment, signifying an advancement in precision oncology and improving survival outcomes in advanced solid tumors.