DNA Analysis Research Articles

Clinical utility of BRCA and ATM mutation status in circulating tumour DNA for treatment selection in advanced pancreatic cancer.

Identification of homologous recombination deficiency (HRD) remains a challenge in advanced pancreatic cancer (APC). We investigated the utility of circulating tumour DNA (ctDNA) profiling in the assessment of BRCA1/2 and ATM mutation status and treatment selection in APC. We analysed clinical and ctDNA data of 702 patients with APC enroled in GOZILA, a ctDNA profiling study using Guardant360. Inactivating BRCA1/2 and ATM mutations were detected in 4.8% (putative germline, 3.7%) and 4.4% (putative germline, 0.9%) of patients, respectively. Objective response (63.2% vs. 16.2%) and PFS (HR 0.55, 95% CI 0.32-0.93) on platinum-containing chemotherapy were significantly better in patients with putative germline BRCA1/2 (gBRCA) mutation than those without. In contrast, putative gBRCA mutation had no impact on the efficacy of gemcitabine plus nab-paclitaxel. In 2 patients treated with platinum-containing therapy, putative BRCA2 reversion mutations were detected. Three of seven patients with somatic BRCA mutations responded to platinum-containing therapy, while only one of four with putative germline ATM mutations did. One-third of somatic ATM mutations were in genomic loci associated with clonal haematopoiesis. Comprehensive ctDNA profiling provides clinically relevant information regarding HRD status. It can be a practical, convenient option for HRD screening in APC.

EDNAqua-Plan—Standardisation Overview for eDNA Sequencing of Aquatic Organisms and the Downstream Data Ecosystem

The eDNAqua-Plan project stands as a beacon of innovation in the biomonitoring of marine and freshwater ecosystems, propelled by the urgent need to integrate DNA-based approaches in aquatic bioassessment and monitoring frameworks. The broad utilisation of cutting-edge environmental DNA (eDNA) and DNA barcoding methodologies is dependent on complete, reliable, and accessible reference DNA sequence data (Rimet et al. 2021). Complete and interoperable metadata is crucial to allow a broad reuse of (e)DNA data and analysis outputs, and for a broader uptake of results by end users. The eDNAqua-Plan project aims to address key limitations to the routine implementation of eDNA-based monitoring methods in Europe by developing plans for federated DNA barcode reference libraries and eDNA data repositories to support DNA-based environmental monitoring. This will ensure a sustainable and reliable infrastructure to underpin its broad use, thereby paving the way for more effective conservation and management strategies. The project is working towards creating a comprehensive overview of standardisation efforts and data workflows, through collaborations with other projects, initiatives and infrastructures for aquatic monitoring across the European Union (EU) and associated countries. We are analysing existing archives (e.g., International Nucleotide Sequence Database Collaboration (INSDC), Barcode of Life Data System (BOLD), Global Biodiversity Information Facility (GBIF), Ocean Biodiversity Information System (OBIS)), portals, and papers to determine current and best practices through the use of questionnaires, manual evaluation of repositories, and machine learning methods (LLMs). This includes an overview of the usage of existing metadata and data standards (e.g., Minimum Information about any (X) Sequence Specifications from the Genomics Standards Consortium (GSC), Darwin Core standard). The results are being integrated by a team of experts in marine and freshwater biomonitoring. With a diverse consortium comprising 18 partner institutions from 11 countries and one international institute, eDNAqua-Plan brings together experts in marine and freshwater monitoring, eDNA analysis, and data science. The collective effort by this consortium will lay the groundwork for the creation of a digital ecosystem of eDNA repositories and an integrated reference library of marine and freshwater species, adhering to FAIR (Findable, Accessible, Interoperable, and Reusable) principles.

Study of Altenia wagneriella (rebel) as a predator associated with Forda marginata from Iran.

The larvae of Altenia spp. and gall aphids are known to feed on plants related to Anacardiaceae. This study documents the aphidophagous habit of Altenia wagneriella, which was verified by molecular techniques, subsequently by the gut dissection test, and direct observation. To identify the moth larvae and adult aphids, two mitochondrial genes, cytochrome c oxidase I (COI) and cytochrome b (Cytb), were amplified by polymerase chain reaction (PCR). Nested PCR with the aphid-specific primer pairs AphidF and AphidR was used to detect aphids in the body of moth larvae. The specificity of the primers was verified by PCR analysis of DNA from moth larvae and adult aphids. The method detected aphids in moth larvae, and a band of approximately 200bp was amplified from moth larvae feeding on aphids. No cross reactions with moth larvae were observed. In the laboratory, all moth larvae feeding on aphids (Forda marginata) were also PCR positive for aphids. Gall-inducing insects are microhabitat engineers that manipulate their host to obtain a better nutrient supply, as well as protection from natural enemies and abiotic factors. This is the first recorded instance worldwide of the carnivorous larva of the moth A. wagneriella acting as an aphid predator, as well as the first record of a host insect for this species. Additionally, it is the first effort to molecularly analyze the predator-prey relationship between the moth larvae and the aphids inside the wild pistachio gall.

Structural insights into the diversity and DNA cleavage mechanism of Fanzor

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

Propagating Terroir Revival in the Negev: How the Wine Industry Can Amplify Its Resilience to Climate Adversity through a Deeper Understanding of Historic Dryland Viticulture

Based on an examination of the outlooks and practices held by past and present Negev desert viticulturists vis-à-vis their environs, the article offers insights into how the wine sector can bolster its response to current climate adversity. The discussion fuses bio-archaeological research on the evolution of ancient grape cultivars (Vitis vinifera) in the Negev with an extensive ethnographic inquiry into the ongoing wine revival in arid southern Israel. Ancient DNA analysis of relic grape material led to the identification of an historical Negev viticulture lineage. The findings provide a temporal framework for contextualizing tphe interrelated anthropological data obtained on contemporary dryland viticulturalists and wine makers in Israel. Since the climate in the Negev has remained relatively stable over the last several millennia, it makes for an apt point of comparison via which to gauge the outlooks held by both historical and modern viticulturalists towards the local landscape. We suggest enacting two evidence-based adjustments to counter ecological instabilities: the holistic revival and applied renewal of historical cultivars, and the attendant agronomic routines that sustained their growth in the past. A more nuanced understanding of the unique terroir arid viticulturalists are operating in, together with the learned incorporation of the hardy genetic traits of age-old cultivars, will benefit wine growers to attune their responses to the array of challenges wrought by climate change on the wine industry. It is therefore our hope that the research methodologies and heritage-oriented horticultural perspectives we present in this paper will prove beneficial to broader wine growing regions (pax-Mediterranean and beyond) who are fortunate to likewise possess their own rich viticultural histories and lineages.

FORENSIC DNA PHENOTYPING FOR CRIMINAL IDENTIFICATION

The purpose of the work – to carry out an analysis of modern achievements andpossibilities of forensic DNA phenotyping to determine the external characteristics ofa person, his biogeographical origin and age in order to facilitate criminal investigations.Conclusions. Forensic DNA Phenotyping (FDP) involves the prediction of a person’sexternal characteristics regarding appearance, biogeographic origin, and age based onDNA samples from a crime scene to support investigations and help to find unknowncriminals who cannot be identified through forensic STR profiling.DNA-based appearance prediction has moved beyond eye, hair and skin color andincluded new features such as eyebrow color, freckles, hair structure, male pattern hairloss and height. DNA-based biogeographic inference of ancestry has progressed fromcontinental ancestry to detecting subcontinental ancestry and resolving models of commonancestry in genetically mixed individuals. DNA-based age estimation is now possible notonly through blood testing, but also through somatic tissues such as saliva and bone, aswell as new markers and tools for analyzing semen. Technological advances have madeit possible to use forensic DNA technology with greatly increased multiplexing capabilityto simultaneously analyze hundreds of DNA predictors using targeted massively parallelsequencing (MPS). Forensically validated MPS-based FDP tools for DNA-based crimescene prediction are used to identify: several features of appearance; multiregional origin;several appearance features together with multiregional origin; age by different tissue types. Despite recent advances that are likely to increase the impact of FDP in criminal casesin the near future, bringing reliable prediction of appearance, ancestry, and age fromcrime scene DNA to the level of detail and accuracy, that forensic scientists may desire,requires further intensive scientific research, along with technical developments andforensic validation.

Mechanism of structure-specific DNA binding by the FANCM branchpoint translocase.

FANCM is a DNA repair protein that recognizes stalled replication forks, and recruits downstream repair factors. FANCM activity is also essential for the survival of cancer cells that utilize the Alternative Lengthening of Telomeres (ALT) mechanism. FANCM efficiently recognizes stalled replication forks in the genome or at telomeres through its strong affinity for branched DNA structures. In this study, we demonstrate that the N-terminal translocase domain drives this specific branched DNA recognition. The Hel2i subdomain within the translocase is crucial for effective substrate engagement and couples DNA binding to catalytic ATP-dependent branch migration. Removal of Hel2i or mutation of key DNA-binding residues within this domain diminished FANCM's affinity for junction DNA and abolished branch migration activity. Importantly, these mutant FANCM variants failed to rescue the cell cycle arrest, telomere-associated replication stress, or lethality of ALT-positive cancer cells depleted of endogenous FANCM. Our results reveal the Hel2i domain is key for FANCM to properly engage DNA substrates, and therefore plays an essential role in its tumour-suppressive functions by restraining the hyperactivation of the ALT pathway.

A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA.

To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement. Through a reproducibility study and several small-scale experiments, the reliability of a custom qPCR assay was established. A reproducibility study illustrated that the multiplex assay produced data comparable to that of previously established bacterial DNA and human DNA qPCR assays. The small-scale experiments showed that common surfaces such as keyboards (6.76 pg/μL), elevator buttons (11.9 pg/μL), cleaning supplies (7.17 pg/μL), and dispensers (16.4 pg/μL) failed to produce human DNA quantities sufficient for quality STR analysis (≥250 pg). However, all tested surfaces produced bacterial DNA quantities suitable for reaching 1 ng of amplified bacterial targets necessary for sequence analysis. In fact, bacterial DNA concentrations down to 10-8 ng/uL produce enough amplified product for sequencing. The newly developed qPCR multiplex tool will allow scientists to make better decisions regarding whether human or bacterial DNA analysis methods can be pursued during forensic or other investigations.

Clinical utility of circulating tumoral dna analysis by multi-gene ngs panels in therapy monitoring of advanced sporadic medullary thyroid carcinoma patients

Clinical utility of circulating tumoral dna analysis by multi-gene ngs panels in therapy monitoring of advanced sporadic medullary thyroid carcinoma patients

Multi-marker DNA metabarcoding for precise species identification in ichthyoplankton samples

Ichthyoplankton monitoring is crucial for stock assessments, offering insights into spawning grounds, stock size, seasons, recruitment, and changes in regional ichthyofauna. This study evaluates the efficiency of multi-marker DNA metabarcoding using mitochondrial cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA gene markers, in comparison to morphology-based methods for fish species identification in ichthyoplankton samples. Two transects with four coastal distance categories were sampled along the southern coast of Portugal, being each sample divided for molecular and morphological analyses. A total of 76 fish species were identified by both approaches, with DNA metabarcoding overperforming morphology—75 versus 11 species-level identifications. Linking species-level DNA identifications with higher taxonomic morphological identifications resolved several uncertainties associated with traditional methods. Multi-marker DNA metabarcoding improved fish species detection by 20–36% compared to using a single marker/amplicon, and identified 38 species in common, reinforcing the validity of our results. PERMANOVA analysis revealed significant differences in species communities based on the primer set employed, transect location, and distance from the coast. Our findings underscore the potential of DNA metabarcoding to assess ichthyoplankton diversity and suggest that its integration into routine surveys could enhance the accuracy and comprehensiveness of fish stock assessments.

Analysis of mitochondrial DNA to estimate the population genetic structure of Tuta absoluta (Meyrick, 1917)

Tuta absoluta (Meyrick) has become a serious menace to the sustainable production of tomatoes in Kenya. Investigating the genetic diversity, geographical distribution, and community structure of T. absoluta from various locations utilizing the Cytochrome c oxidase I (COI) of the mtDNA gene information available on NCBI databases. Based on the COI of mtDNA, the haplotype analysis revealed six haplotypes, which displayed low nucleotide (π = 0.00021) diversity. In contrast, haplotype diversity (Hd) was 0.05, and the number of segregating sites (S) was 13. The result showed that the tomato leaf miner population from Central Kenya had high variability in haplotype supported by the haplotype network. Tajima (D) and Fu's Fs were found to be negative (P greater than 0.05), whereas the overall FST value was 0.00077 (P greater than 0.001). In conclusion, genetic analysis showed there was no high variation amongst population from different locations.

Clinical value of sequential circulating tumor DNA analysis using next-generation sequencing and epigenetic modifications for guiding thermal ablation for colorectal cancer metastases: a prospective study.

While thermal ablation is now a standard treatment option for oligometastatic colorectal cancer patients, selecting those who will benefit most from locoregional therapies remains challenging. This proof-of-concept study is the first to assess the feasibility of routine testing of ctDNA before and after thermal ablation with curative intent, analyzed by next-generation sequencing (NGS) and methylation specific digital droplet PCR (ddPCR). Our prospective study primary objective was to assess the prognostic value of ctDNA before thermal ablation. This single-center prospective study from November 2021 to June 2022 included colorectal cancer patients referred for curative-intent thermal ablation. Cell-free DNA was tested at different time points by next-generation sequencing and detection of WIF1 and NPY genes hypermethylation using ddPCR. The ctDNA was considered positive if either a tumor mutation or hypermethylation was detected; recurrence-free survival was used as the primary endpoint. The study enrolled 15 patients, and a total of 60 samples were analyzed. The median follow-up after ablation was 316days, and median recurrence-free survival was 250days. CtDNA was positive for 33% of the samples collected during the first 24h. The hazard ratio for progression according to the presence of baseline circulating tumor DNA was estimated at 0.14 (CI 95%: 0.03-0.65, p = 0.019). The dynamics are provided, and patients with no recurrence were all negative at H24 for ctDNA. This study shows the feasibility of routine testing of ctDNA before and after thermal ablation with curative intent. We report that circulating tumor DNA is detectable in patients with low tumor burden using 2 techniques. This study emphasizes the potential of ctDNA for discerning patients who are likely to benefit from thermal ablation from those who may not, which could shape future referrals. The dynamics of ctDNA before and after ablation shed light on the need for further research and larger studies.

Label-Free Detection of DNA Base Mutation and Hybridized DNA by an Electrostatically Modified 3D Plasmonic Array.

Surface-enhanced Raman scattering (SERS) represents a promising avenue for DNA detection as it offers intrinsic chemical insights with high sensitivity compared to conventional methods. However, label-free and quantitative detection of unmodified DNA by SERS remains a major challenge in DNA analysis. To overcome this challenge, we propose a positively charged plasmonic nanosurface for DNA capture and quantitative analysis. Highly sensitive and uniform SERS enhancement was realized by a three-dimensional plasmonic array supporting well-designed hybrid plasmonic modes. Subsequently, the plasmonic array was modified with an electrostatically functionalized PDDA (poly(diene-dimethylammonium-chloride)) self-assembled monolayer in a single step. The effectiveness of the resulting PDDA-SERS substrate was demonstrated by the label-free and quantitative detection of base content and base mutation in hybridized DNA. The PDDA-SERS substrate provides a robust platform for SERS analysis not only of DNA but also of other electronegative analytes.

Genomic and epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse survival in lethal prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSIs) is often lethal. Liquid biopsy biomarkers for this deadly form of disease remain under investigation, and underpinning mechanisms remain ill-understood. We applied targeted cell-free DNA sequencing to 126 mCRPC patients from three academic cancer centers, and separately performed genome-wide cell-free DNA methylation sequencing on 43 plasma samples collected prior to the initiation of first-line ARSI treatment. To analyze the genome-wide sequencing data, we performed nucleosome-positioning and differential methylated region analysis. We additionally analyzed single-cell and bulk RNA sequencing data from 14 and 80 mCRPC patients, respectively, to develop and validate a stem-like signature, which we inferred from cell-free DNA. Targeted cell-free DNA sequencing detected AR/enhancer alterations prior to first-line ARSIs which correlated with significantly worse PFS (p = 0.01; HR = 2.12) and OS (p = 0.02; HR = 2.48). Plasma methylome analysis revealed that AR/enhancer lethal mCRPC patients have significantly higher promoter-level hypomethylation than AR/enhancer wild-type mCRPC patients (p < 0.0001). Moreover, gene ontology and CytoTRACE analysis of nucleosomally more accessible transcription factors in cell-free DNA revealed enrichment for stemness-associated transcription factors in lethal mCRPC patients. The resulting stemness signature was then validated in a completely held-out cohort of 80 mCRPC patients profiled by tumor RNA sequencing. We analyzed a total of 220 mCRPC patients, validated the importance of cell-free AR/enhancer alterations as a prognostic biomarker in lethal mCRPC and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness.

Identification and Characterization of Extrachromosomal Circular DNA in Slimming Grass Carp.

Slimming grass carp is a commercial variety with good body form and meat quality, which is cultured by starving common grass carp in a clean flowing water environment. Compared to common grass carp, slimming grass carp has a far higher economic value. Until now, no molecular study has concentrated on the regulation mechanism of the muscle characteristics of slimming grass carp. This study first reported the gene expression profile of the muscle characteristics of slimming grass carp based on the level of extrachromosomal circular DNAs (eccDNAs). EccDNAs are double-stranded circular DNAs derived from genomic DNAs and play crucial roles in the functional regulation of a wide range of biological processes, none of which have been shown to occur in fish. Here, muscle eccDNAs from slimming grass carp and common grass carp were both generally sequenced, and the information, as well as the expression profile of eccDNAs, were compared and analysed. The findings reveal that 82,238 and 25,857 eccDNAs were detected from slimming grass carp and common grass carp, respectively. The length distribution of eccDNAs was in the range of 1~1000 bp, with two peaks at about 200 bp and 400 bp. When the expression profiles of eccDNAs between slimming grass carp and common grass carp were compared, 3523 up-regulated and 175 down-regulated eccDNAs were found. Enrichment analysis showed that these eccDNA genes were correlated with cellular structure and response, cell immunology, enzyme activity, etc. Certain differentially expressed eccDNAs involved in muscle characteristics were detected, which include myosin heavy chain, myosin light chain, muscle segment homeobox C, calsequestrin, calmodulin, etc., among which the majority of genes were linked to muscle structure and contraction. This indicates that during the process of cultivating from common grass carp to slimming grass carp, the treatment primarily affected muscle structure and contraction, making the meat quality of slimming grass carp different from that of common grass carp. This result provides molecular evidence and new insights by which to elucidate the regulating mechanism of muscle phenotypic characterisation in slimming grass carp and other fish.

Sex estimation using long bones in the largest burial site of the Copper Age: Linear discriminant analysis and random forest

Sex estimation of the individuals in a sample is fundamental for any bioarchaeological study to define a particular demographic assemblage or to classify isolated remains. Long bones are an excellent alternative for sex estimation when the most dimorphic anatomical parts are not preserved or are highly altered. Here we propose a set of discriminant functions and classification models to estimate the sex of prehistoric individuals using linear discriminant analysis and machine learning approaches. Different osteometric variables were taken from the humeri, ulnae, radii, femurs and tibias of a sample of 109 articulated skeletons buried in the collective tomb of Camino del Molino (Region of Murcia, SE-Spain), dated to the 3rd millennium BC. Sex was estimated based on standard anthropological methods and ancient DNA analysis of a control sample. Fifty-two discriminant functions with prediction thresholds higher than 0.8 on the ROC curve were obtained using independent (22) and combined variables (30). The best LDA models for sex prediction were those based on proximal epiphyseal widths or their combination with other variables, reaching values close to 0.98 on the ROC curve. The random forest-based model obtained an accuracy of 0.94 and confirmed the importance of epiphyseal widths in sex classification. This analysis is more comprehensive than univariate LDA, as it allows for ranking the importance of bones in sex discrimination and considers correlations between long bones rather than treating them as independent observations. In contrast, applying LDA to each bone makes it easier to predict the sex of other coeval collections that do not have such a complete sample. This work aims to overcome the scarcity of methods that can be applied to sex estimation of the large volume of isolated remains from Camino del Molino and for other Mediterranean skeletal series from the Late Prehistory with high biological affinity and that share similar environmental conditions.

Timber Tracking of Jacaranda copaia from the Amazon Forest Using DNA Fingerprinting

Timber Tracking of Jacaranda copaia from the Amazon Forest Using DNA Fingerprinting

Assessment of Correlation between Genetic Taste Perception Hormonal Fingerprint and Dental Caries Incidence in Schoolgoing Children: An In Vivo Study

Assessment of Correlation between Genetic Taste Perception Hormonal Fingerprint and Dental Caries Incidence in Schoolgoing Children: An In Vivo Study

Facultative parthenogenesis discovered for the first time in Jamaican boa (Chilabothrus subflavus) using novel microsatellite markers.

The mode of reproduction most often seen in snakes is sexual, but studies have noted facultative parthenogenesis in at least six families. Here, we provide evidence for the first observed case of facultative parthenogenesis in a captive Jamaican boa (Chilabothrus subflavus). A 7-year-old female Jamaican boa, isolated since birth, was found to have produced a litter of 15 offspring. To provide molecular DNA evidence of parthenogenesis, 13 new microsatellite loci were isolated in the species. All offspring were found to be homozygous at each locus and only possess alleles found in the dam, implicating that they were born from asexual reproduction. Several developmental abnormalities, including stillbirths and spinal deformities, were noted in the litter which may be explained by their increased level of homozygosity. To preserve genetic diversity in the captive population, research should be conducted to understand the prevalence of this mode of reproduction and to guide future management decisions of this IUCN listed Vulnerable species.

New Variants in the Chloroplast Genome Sequence of Two Colombian Individuals of the Cedar Timber Species (Cedrela odorata L.), Using Long-Read Oxford Nanopore Technology

The plant species Cedrela odorata has been largely exploited in the timber industry due to the high demand for its wood. Therefore, C. odorata has been considered a vulnerable species since 1994, under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). C. odorata is a key timber species included in the management and conservation plans for the Amazon and Central American rainforests. These plans include the development of genetic and genomic resources to study local populations of the species in Colombia. In this study, two novel chloroplast (cp) genomes were assembled and annotated using the MinION long-read sequencing technology. The new cp genomes were screened for sequence variants (SVs), and a total of 16 SNPs were identified, presumably unique to populations from the Amazon region in Colombia. Comparative genomics with other reported cp genomes from different populations of C. odorata support the hypothesis of intraspecific diversity associated with their place of origin. These cp genome sequences of C. odorata from Colombian individuals represent valuable genomic resources for the species, suitable for identifying novel DNA fingerprinting and barcoding applications.