DNA Analysis Research Articles

Validation of an eDNA‐based workflow for monitoring inter‐ and intra‐specific CytB haplotype diversity of alpine amphibians

AbstractEnvironmental DNA (eDNA) analysis is a promising tool for monitoring wild animal populations and, more recently, their genetic variability. In this study, we used the mitochondrial Cytochrome B gene to develop and apply new eDNA metabarcoding assays targeting amphibian families and genera in order to estimate both inter‐ and intraspecific genetic diversity. We designed and tested seven new primer pairs (a) in silico against an amphibian reference database based on the target genera; (b) in vitro on tissue samples of the target species; and (c) in situ on water samples from 38 wetlands in the Province of Trento (Italy). Overall, most target species were amplified successfully, although some markers also amplified non‐target amphibian species. In addition, to complete the workflow, we compared the performance of three different bioinformatic pipelines (namely, MICCA with VSEARCH, and OBITools using ecotag or metabinkit), in retrieving reads and exact sequence variants from the metabarcoding datasets. Overall, the MICCA based pipeline retrieved more reads, but less putative haplotypes of amphibians. After comparing these sequences with previously known haplotypes from tissue‐based studies, when the aim is to both decrease the probability of detecting false haplotypes and retrieve the highest number of reads, we suggest using MICCA+VSEARCH, unless a direct comparison with tissue‐based genetic data is possible.

118P Comparative analysis of DNA and RNA-based NGS for detecting MET exon 14 skipping mutation in pan-solid tumor samples

118P Comparative analysis of DNA and RNA-based NGS for detecting MET exon 14 skipping mutation in pan-solid tumor samples

DNA fingerprinting of yeast (Saccharomyces cerevisiae) strains by using molecular marker

DNA fingerprinting of yeast (Saccharomyces cerevisiae) strains by using molecular marker

Decreased accuracy of forensic DNA mixture analysis for groups with lower genetic diversity

Decreased accuracy of forensic DNA mixture analysis for groups with lower genetic diversity

Single-cell multi-modal chromatin profiles revealing epigenetic regulations of cells in hepatocellular carcinoma.

Various epigenetic regulations systematically govern gene expression in cells involving various biological processes. Dysregulation of the epigenome leads to aberrant transcriptional programs and subsequently results in diseases, such as cancer. Therefore, comprehensive profiling epigenomics is essential for exploring the mechanisms underlying gene expression regulation during development and disease. In this study, we developed single-cell chromatin proteins and accessibility tagmentation (scCPA-Tag), a multi-modal single-cell epigenetic profile capturing technique based on barcoded Tn5 transposases and a droplet microfluidics platform. scCPA-Tag enables the simultaneous capture of DNA profiles of histone modification and chromatin accessibility in the same cell. By applying scCPA-Tag to K562 cells and a hepatocellular carcinoma (HCC) sample, we found that the silence of several chromatin-accessible genes can be attributed to lysine-27-trimethylation of the histone H3 tail (H3K27me3) modification. We characterized the epigenetic features of the tumour cells and different immune cell types in the HCC tumour tissue by scCPA-Tag. Besides, a tumour cell subtype (C2) with more aggressive features was identified and characterized by high chromatin accessibility and a lower abundance of H3K27me3 on tumour-promoting genes. Our multi-modal scCPA-Tag provides a comprehensive approach for exploring the epigenetic landscapes of heterogeneous cell types and revealing the mechanisms of gene expression regulation during developmental and pathological processes at the single-cell level. scCPA-Tag offers a highly efficient and high throughput technique to simultaneously profile histone modification and chromatin accessibility within a single cell. scCPA-Tag enables to uncover multiple epigenetic modification features of cellular compositions within tumor tissues. scCPA-Tag facilitates the exploration of the epigenetic landscapes of heterogeneous cell types and provides the mechanisms governing gene expression regulation.

Moderate evidence for the sex‐dependent effect of poisoning on adult survival in a long‐lived raptor species

AbstractSurvival rate is usually the greatest contributor to population growth in long‐lived species, and its accurate estimation along with the evaluation of the factors influencing it is therefore essential for effective conservation. Here, we studied the survival of breeding eastern imperial eagles Aquila heliaca in Hungary between 2011 and 2022 and investigated the effect of poisoning, the leading known anthropogenic cause of mortality. We used the Cormack‐Jolly‐Seber mark‐recapture model to estimate annual apparent survival and encounter probabilities based on the capture histories of 208 males and 411 females. We obtained encounter data from the DNA profiles of shed feathers collected at the nest sites, which we also supplemented with presences inferred from parentage analysis. The most supported model estimated a constant 91.6% annual survival over the study period, but models including the effect of sex and poisoning rate on survival had similar support. Sex difference in survival was less than 1% on average, but the survival of males decreased more with poisoning rate than the survival of females. However, due to smaller encounter probabilities, the estimates for males were less precise compared to females. Males may be more at risk from poisoning than females not only due to their more active foraging behaviour during incubation and chick‐rearing but also due to their smaller body size. Apart from providing direct practical information for the conservation management of imperial eagles, our results also highlight the importance of long‐term studies for estimating population parameters of birds of prey.

Estimating the sensitivity of genomic newborn screening for treatable inherited metabolic disorders

PurposeOver 30 research groups and companies are exploring newborn screening using genomic sequencing (NBSeq), but the sensitivity of this approach is not well understood. MethodsWe identified individuals with treatable inherited metabolic disorders (IMDs) and ascertained the proportion whose DNA analysis revealed explanatory deleterious variants (EDVs). We examined variables associated with EDV detection and estimated the sensitivity of DNA-first NBSeq. We further predicted the annual rate of true-positive and false-negative NBSeq results in the United States for several conditions on the Recommended Uniform Screening Panel. ResultsWe identified 635 individuals with 80 unique IMDs. In univariate analyses, Black race (OR = 0.37, 95% CI: 0.16-0.89, P = .02) and public insurance (OR = 0.60, 95% CI: 0.39-0.91, P = .02) were less likely to be associated with finding EDVs. Had all individuals been screened with NBSeq, the sensitivity would have been 80.3%. We estimated that between 0 and 649.9 cases of Recommended Uniform Screening Panel IMDs would be missed annually by NBSeq in the United States. ConclusionThe overall sensitivity of NBSeq for treatable IMDs is estimated at 80.3%. That sensitivity will likely be lower for Black infants and those who are on public insurance.

26P Multi-feature cell free DNA analysis and ensemble machine learning for early detection of cancer

26P Multi-feature cell free DNA analysis and ensemble machine learning for early detection of cancer

562P Acquired genomic alterations on first-line chemotherapy (CT) + cetuximab in advanced colorectal cancer (mCRC): Circulating tumor (ct)DNA analysis of the randomized phase II trial TIME-PRODIGE-28

562P Acquired genomic alterations on first-line chemotherapy (CT) + cetuximab in advanced colorectal cancer (mCRC): Circulating tumor (ct)DNA analysis of the randomized phase II trial TIME-PRODIGE-28

Rapid molecular profiling utilising minimal quantities of endobronchial ultrasound-guided aspirates for the detection of Epidermal Growth Factor Receptor, KRAS, ALK, ROS1, RET, NTRK and MET gene alterations from patients with non-small-cell lung carcinomas on the Biocartis Idylla™ platform.

Comprehensive molecular analysis for patients with non-small-cell lung carcinoma (NSCLC) is essential for managing modern targeted therapies. This study sought to establish the feasibility of utilising real-time PCR to perform rapid and comprehensive profiling on minimal amounts of endobronchial ultrasound-guided (EBUS) aspirates as a fast, tissue-sparing route of predictive profiling. A volume of 500 μL of EBUS aspirate and fixative from patients with NSCLC was decanted, and 80 μL (<1% of total specimen received) was utilised for analysis. Biocartis Idylla™ cartridges for epidermal growth factor receptor (EGFR) mutations, KRAS mutations and a GeneFusion cartridge (ALK, ROS1, RET, NTRK1/2/3 rearrangements & MET 14 exon skipping) were analysed for each case to provide molecular data on the main clinically relevant targets as per UK guidelines. A total of 62 cases were included; all of which had successful DNA analysis (EGFR and KRAS cartridges). RNA analysis (GeneFusion cartridge) was successful for 42 of 51 (82%) with initial approach, with 11 of 11 (100%) achieving a successful result with modified protocol. In all, 23 KRAS mutations (37%), 5 EGFR mutations (8%) and 1 ROS fusion (2%) were identified. Average time from specimen receipt to molecular read-out was 5 h. Real-time PCR utilising the Idylla™ platform is rapid, utilises minimal amounts of tissue and provides accurate results. We propose this is a useful ancillary method to utilise alongside next-generation sequencing (NGS) in cases of urgent clinical requirement or EBUS aspirates with inadequate quantities of tissue for NGS.

112P Reporting of molecular test results from cell-free DNA analyses: Expert consensus recommendations from the 2023 European Liquid Biopsy Society ctDNA workshop

112P Reporting of molecular test results from cell-free DNA analyses: Expert consensus recommendations from the 2023 European Liquid Biopsy Society ctDNA workshop

Long-Lasting Response to Lorlatinib in Patients with ALK-Driven Relapsed or Refractory Neuroblastoma Monitored with Circulating Tumor DNA Analysis.

We present five patients with ALK-driven relapsed or refractory neuroblastoma treated with lorlatinib as monotherapy. All patients responded to treatment, and four of them were alive after 3 to 5 years of follow-up. We performed longitudinal ctDNA analysis with ultra-deep sequencing of the ALK tyrosine kinase domain. We conclude that ctDNA analysis may guide treatment decisions in ALK-driven neuroblastoma, also when the disease is undetectable using standard clinical methods.

Environmental DNA as Early Warning for Alien Species in Mediterranean Coastal Lagoons: Implications for Conservation and Management

Non-indigenous species (NIS) introduction notoriously threatens the Mediterranean Sea. In addition, Mediterranean coastal lagoons play a crucial role as nurseries for marine species, which new NIS arrivals can threaten. Therefore, monitoring and early warning of NIS presence is essential in preserving biodiversity. An innovative technique for rapid and accurate species identification and biodiversity screening is the application of environmental DNA (eDNA) metabarcoding. In this research, different Penaeidae (Arthropoda, Crustacea, Decapoda) NIS specimens were collected from a Mediterranean coastal lagoon after an early warning about a potentially invasive NIS arising from next-generation sequencing data. DNA barcoding of the DNA extracted from tissue samples and amplified with specifically designed primer pairs led to the recognition of Penaeus aztecus in this NATURA 2000 protected ecosystem for the first time. DNA barcoding from DNA isolated from the water where the living specimens were stored further validated the possibility of identifying P. aztecus starting from eDNA. This approach demonstrated the validity of environmental DNA analysis in the early screening of potentially invasive NIS presence in Mediterranean protected areas and ecosystems. This work describes an applicative example of the efficacy in improving the biomonitoring of lagoon ecosystems using molecular tools and it represents a guideline for the validation of eDNA metabarcoding data for the presence of potentially invasive species.

1933P Utility of circulating tumoral DNA analysis by multi-gene NGS panels in tracking therapy progression of advanced sporadic medullary thyroid carcinoma

1933P Utility of circulating tumoral DNA analysis by multi-gene NGS panels in tracking therapy progression of advanced sporadic medullary thyroid carcinoma

Retos Actuales de la Medicina Genomica

Genomic Medicine is the application to clinical practice and research of knowledge about the genetic bases of human health and disease. It began in the 1950s and developed throughout the 21st century, starting with the publication of the sequencing of the Human Genome. Its objective is to obtain more precise and earlier diagnoses, prevent diseases, adapt treatments according to individual genetic information and improve public health programs. Advances in DNA sequencing and analysis have made it possible, identifying genetic variants associated with diseases and responses to treatments. These have led to practical applications such as risk prediction and treatment personalization. In 2024, Genomic Medicine has been integrated into routine healthcare and new frontiers such as gene therapy and gene editing are being explored. Despite the advances, Genomic Medicine faces technical, scientific, ethical-legal, organizational and sustainability challenges. However, with interdisciplinary collaboration, these challenges can be overcome to improve human health and well-being.

Optimizing the microbial community composition in anaerobic digesters to improve biogas yields from food waste

Methods: Food waste collected from local restaurants and households in New York city will be used in this study. The food waste will be characterized to analyze its composition. Batch anaerobic digesters will be inoculated with microbial communities from existing food waste digesters. DNA analysis using 16S rRNA gene sequencing will be done to show the initial microbial consortia. Various perturbations will be applied to the digesters including changes in temperature, pH, feeding rates, mixing intensities to select for microbial populations yielding higher biogas production. Biogas production will be monitored over time. DNA analysis will be repeated after perturbation to analyze changes in the microbial community structure. Results: Preliminary results show Food waste characterized was found to contain 35% proteins, 45% carbohydrates and 20% fats. The initial microbial consortia in the digesters was dominated by bacteria from the genera Clostridium, Bacteroides and Methanothermobacter. Increasing feeding rates led to a 40% increase in biogas yields. Microbial analysis indicated a shift in populations with Clostridium becoming the most abundant genus. Decreasing pH from 7 to 6.5 further enhanced biogas by 25% with Clostridium and Syntrophomonas becoming dominant. Discussion: The study demonstrates that optimizing operational conditions can effectively manipulate the microbial communities in anaerobic digesters processing food waste. By applying selective pressures, populations better suited to degrade the local food waste and produce higher methane yields can be enriched. Further experiments will aim to construct stable, optimized consortia through controlled perturbation for robust and efficient food waste digestion. The approach holds promise for improving biogas production from food waste globally

Loss of Heterozygosity and Mutations in the RAS-ERK Pathway Genes in Tumor Cells of Various Loci in Multiple Myeloma.

Multiple myeloma (MM) is a disease characterized by spatiotemporal heterogeneity of tumor clones. Different genetic aberrations can be observed simultaneously in tumor cells from different loci, and as the disease progresses, new subclones may appear. The role of liquid biopsy, which is based on the analysis of tumor DNA circulating in the blood plasma, continues to be explored in MM. Here, we present an analysis of the STR profiles and mutation status of the KRAS, NRAS, and BRAF genes, evaluated in plasma free circulating tumor DNA (ctDNA), CD138+ bone marrow cells, and plasmacytomas. The prospective single-center study included 97 patients, with a median age of 55 years. Of these, 94 had newly diagnosed symptomatic MM, and three had primary plasma cell leukemia. It should be noted that if mutations were detected only in ctDNA, "non-classical" codons were more often affected. A variety of adverse laboratory and clinical factors have been associated with the detection of rare KRAS or NRAS gene mutations in bone marrow or ctDNA, suggesting that these mutations may be factors of an unfavorable prognosis for MM. Liquid biopsy studies provide undeniable fundamental information about tumor heterogeneity and clonal evolution in MM. Moreover, we focus on using liquid biopsy to identify new high-risk factors for MM.

Detection of reproductive interference between closely related Salvia species with small-scale separated distributions by multifaceted pollination and molecular analyses

Reproductive interference, an interspecific interaction in reproductive process that exerts an adverse effect, has gained attention as a contributing factor in promoting exclusive distributions between closely related species. However, detailed studies on the possibility of reproductive interference between native plants are still lacking, presumably because strong reproductive interference can rapidly realize exclusive distributions, leaving the two species apparently independent. Salvia japonica and S. lutescens are found in separate localities at a small scale, although their distributions overlap at a large scale. We investigated the possibility of reproductive interference between them through field surveys, hand-pollination experiments, evaluation of hybrid fertility, cpDNA and nrDNA genotyping, and genome-wide DNA analysis. The field survey results did not reveal apparent negative interaction in competition for pollinator services. Mixed pollination with conspecific pollen and counterpart pollen reduced seed set in S. japonica, and hybrid progeny produced by mixed pollination were less than 20% as fertile compared to the pure species. The DNA genotyping results suggested the possibility of hybridization where their distributions overlap, and the genome-wide DNA analysis results showed clear genetic differentiation between the two species as well as the existence of hybrids. These results suggest that bi-directional reproductive interference between S. japonica and S. lutescens may have led to their present separated distributions at a small scale.

Comparison of commercial DNA kits for allergen detection of celery in food matrices

For correct allergen risk management by industry, retail and food safety authorities, sensitive and reliable fast allergen detection methods are required, even more when precautionary allergen labelling based on reference doses will be implemented in legislation.This study aimed to perform a comparative assessment of three commercially available quantitative or qualitative test kits, for DNA analysis of celery in food products. Five product groups, representing different sectors of the AOAC food-matrix triangle, being (plant-based) meat products, snacks, sauces, dried herbs and spices, and smoothies, were identified to potentially contain celery. From each group, blank and incurred (labelled to contain celery) food products were selected, of which the blank food products were additionally spiked with low protein levels of celery prior to qPCR assessment.Results show that the assessed test kits perform according to their specifications, however, a clear influence of the matrix on the detection ability of celery was observed. In addition, quantification of the amount of celery in the different food products showed to be challenging in all food product groups using the two quantification kits.

Methylation-Associated Nucleosomal Patterns of Cell-Free DNA in Cancer Patients and Pregnant Women.

Cell-free DNA (cfDNA) analysis offers an attractive noninvasive means of detecting and monitoring diseases. cfDNA cleavage patterns within a short range (e.g., 11 nucleotides) have been reported to correlate with cytosine-phosphate-guanine (CpG) methylation, allowing fragmentomics-based methylation analysis (FRAGMA). Here, we adopted FRAGMA to the extended region harboring multiple nucleosomes, termed FRAGMAXR. We profiled cfDNA nucleosomal patterns over the genomic regions from -800 to 800 bp surrounding differentially methylated CpG sites, harboring approximately 8 nucleosomes, referred to as CpG-associated cfDNA nucleosomal patterns. Such nucleosomal patterns were analyzed by FRAGMAXR in cancer patients and pregnant women. We identified distinct cfDNA nucleosomal patterns around differentially methylated CpG sites. Compared with subjects without cancer, patients with hepatocellular carcinoma (HCC) showed reduced amplitude of nucleosomal patterns, with a gradual decrease over tumor stages. Nucleosomal patterns associated with differentially methylated CpG sites could be used to train a machine learning model, resulting in the detection of HCC patients with an area under the receiver operating characteristic curve of 0.93. We further demonstrated the feasibility of multicancer detection using a dataset comprising lung, breast, and ovarian cancers. The tissue-of-origin analysis of plasma cfDNA from pregnant women and cancer patients revealed that the placental DNA and tumoral DNA contributions deduced by FRAGMAXR correlated well with values measured using genetic variants (Pearson r: 0.85 and 0.94, respectively). CpG-associated cfDNA nucleosomal patterns of cfDNA molecules are influenced by DNA methylation and might be useful for biomarker developments for cancer liquid biopsy and noninvasive prenatal testing.