DNA Analysis Research Articles

Data-driven fingerprint nanoelectromechanical mass spectrometry

Fingerprint analysis is a ubiquitous tool for pattern recognition with applications spanning from geolocation and DNA analysis to facial recognition and forensic identification. Central to its utility is the ability to provide accurate identification without an a priori mathematical model for the pattern. We report a data-driven fingerprint approach for nanoelectromechanical systems mass spectrometry that enables mass measurements of particles and molecules using complex, uncharacterized nanoelectromechanical devices of arbitrary specification. Nanoelectromechanical systems mass spectrometry is based on the frequency shifts of the nanoelectromechanical device vibrational modes that are induced by analyte adsorption. The sequence of frequency shifts constitutes a fingerprint of this adsorption, which is directly amenable to pattern matching. Two current requirements of nanoelectromechanical-based mass spectrometry are: (1) a priori knowledge or measurement of the device mode-shapes, and (2) a mode-shape-based model that connects the induced modal frequency shifts to mass adsorption. This may not be possible for advanced nanoelectromechanical devices with three-dimensional mode-shapes and nanometer-sized features. The advance reported here eliminates this impediment, thereby allowing device designs of arbitrary specification and size to be employed. This enables the use of advanced nanoelectromechanical devices with complex vibrational modes, which offer unprecedented prospects for attaining the ultimate detection limits of nanoelectromechanical mass spectrometry.

Who threw that stone? A study on DNA transfer

Contact or touch DNA traces from stones account for around 5 % of all crime scene-related swab samples analysed in our department. These traces are often used to identify perpetrators in cases such as burglary, when a stone is used as a tool to break a window or in cases of property damage during riots. Provided that a DNA profile can be obtained in such a case, questions may arise in court regarding the possibilities of DNA transfer onto the stone. Was the subject's DNA indeed transferred onto the stone while it was being used for the crime, or was it already present as background DNA? Alternatively, could it have been transferred by other means, such as by handing over the stone to someone else who then threw it, or by touching it during an attempt to prevent someone else from throwing it? This study focused on two scenarios: experiments involving different participants throwing various stones and a handover scenario where one person touched the stone and another person threw it. We observed that the amount of DNA transferred/detected on the stone is mainly dependent on the individual handling it rather than on the properties of the stone itself or on the order in which the stones are thrown. In the handover scenario, the person who first touched the stone was found to be the main contributor to the trace as often as the person who eventually threw the stone. Our findings therefore confirm that no conclusions can be drawn about the way of interaction with the stones based solely on the obtained DNA profiles.

Revisiting the Briggs Ancient DNA Damage Model: A Fast Maximum Likelihood Method to Estimate Post-Mortem Damage.

One essential initial step in the analysis of ancient DNA is to authenticate that the DNA sequencing reads are actually from ancient DNA. This is done by assessing if the reads exhibit typical characteristics of post-mortem damage (PMD), including cytosine deamination and nicks. We present a novel statistical method implemented in a fast multithreaded programme, ngsBriggs that enables rapid quantification of PMD by estimation of the Briggs ancient damage model parameters (Briggs parameters). Using a multinomial model with maximum likelihood fit, ngsBriggs accurately estimates the parameters of the Briggs model, quantifying the PMD signal from single and double-stranded DNA regions. We extend the original Briggs model to capture PMD signals for contemporary sequencing platforms and show that ngsBriggs accurately estimates the Briggs parameters across a variety of contamination levels. Classification of reads into ancient or modern reads, for the purpose of decontamination, is significantly more accurate using ngsBriggs than using other methods available. Furthermore, ngsBriggs is substantially faster than other state-of-the-art methods. ngsBriggs offers a practical and accurate method for researchers seeking to authenticate ancient DNA and improve the quality of their data.

Impact of periodontal therapy on oral bacterial composition in individuals diagnosed with advanced periodontal disease.

Introduction. Negative changes in the microbial composition have been extensively studied in individuals with periodontal disease.Gap Statement. The changes in the oral microbiota after treating this disease are still unknown.Aim. We sought to elucidate the distinctive traits of salivary microbiota in individuals displaying healthy gums and those with severe periodontitis (SP) and examine the influence of periodontal therapy.Methodology. Periodontal pocket depths were examined to determine disease severity. The presence and quantity of oral Helicobacter pylori (associated with periodontal disease) were determined. Sequencing of 16S ribosomal DNA and bioinformatic analyses were performed to assess oral bacterial compositions in patients.Results. Sequencing analysis of 16S ribosomal DNA revealed a significant reduction in the abundance of operational taxonomic units (OTUs) and Chao1 and Abundance coverage-based estimator(ACE) indices in the oral cavities of individuals with SP compared to those of the healthy controls. However, these parameters showed significant recovery after appropriate treatment severe periodontitis after treatment (TSP). Additionally, the levels of harmful Bacillales and Spirochetes significantly increased, whereas the presence of beneficial Euryarchaeota significantly decreased in the SP group. The TSP group exhibited considerably augmented abundances of Burkholderiaceae and Veillonella, while noteworthy reductions in the pathogenic microbiota (Clostridia, Fusobacteria and Spirochaetes) were noticed compared to those of the SP group. Functionally, these modified OTUs were extensively implicated in 41 metabolic pathways.Conclusion. Our study demonstrates that nonsurgical periodontal therapy can effectively reduce the diversity of the oral microbiota, thereby potentially enhancing the treatment efficacy in patients with periodontal disease.

Expanding the Role of Molecular Genetic Expertise in Crime Detection and Investigation

Molecular genetic testing using DNA plays a crucial role in the field of justice. Beyond the analysis of genomic DNA sequences, advanced methods such as mitochondrial DNA analysis, epigenetic marker identification, and RNA profiling are being developed. A particularly promising approach is forensic phenotyping, which involves predicting human characteristics from DNA samples, including appearance, biogeographic origin, and age.

Tendons and table salt: A recipe to preserve human DNA

For disaster victim identification, hundreds of samples of human remains have to be extracted from human bodies rapidly for later DNA analysis, often in situation with high humidity, high temperature and no access to cooling facilities. Genetic typing of these samples may be challenging due to degradation of DNA. Until today, bone and teeth are the preferred biological matrix for genetic identification of decomposed human remains. However, processing these samples is time and cost consuming. To address the challenges associated with workload, finances, and sample preservation in the genetic identification of unknown dead bodies, we tested 44 hand and foot tendons from deceased with advanced post mortem changes as alternative DNA material. In addition, we examined commercially available table salt as potential preservation method with 10 tendons over a period of 12 months. The parameters external appearance, DNA quantity and quality have been analysed. Overall, 39 of 44 tendons examined yielded valuable DNA profiles with at least ten autosomal STR loci. Storage in table salt not only increased DNA yield but also resulted in high quality STR profiles of the preserved tendons throughout the study period of 12 months compared to storage without salt. According to the results of this study, we recommend hand and foot tendons as alternative DNA material from bodies with advanced post mortem changes. We particularly recommend commercially available table salt as a storage medium for the preservation of DNA samples. Under field conditions without cooling facilities and the need to store samples prior to DNA analysis table salt is a good storage medium: it preserves DNA and is easily available.

Chromosomal Localization and Diversity Analysis of 5S and 18S Ribosomal DNA in 13 Species from the Genus Ipomoea

Background: Sweet potato (Ipomoea batatas (L.) Lam.), a key global root crop, faces challenges due to its narrow genetic background. This issue can be addressed by utilizing the diverse genetic resources of sweet potato’s wild relatives, which are invaluable for its genetic improvement. Methods: The morphological differences in leaves, stems, and roots among 13 Ipomoea species were observed and compared. Chromosome numbers were determined by examining metaphase cells from root tips. Fluorescence in situ hybridization (FISH) was used to identify the number of 5S and 18S rDNA sites in these species. PCR amplification was performed for both 5S and 18S rDNA, and phylogenetic relationships among the species were analyzed based on the sequences of 18S rDNA. Results: Three species were found to have enlarged roots among the 13 Ipomoea species. Chromosome analysis revealed that I. batatas had 90 chromosomes, Ipomoea pes-tigridis had 28 chromosomes, while the remaining species possessed 30 chromosomes. Detection of rDNA sites in the 13 species showed two distinct 5S rDNA site patterns and six 18S rDNA site patterns in the 12 diploid species. These rDNA sites occurred in pairs, except for the seven 18S rDNA sites observed in Ipomoea digitata. PCR amplification of 5S rDNA identified four distinct patterns, while 18S rDNA showed only a single pattern across the species. Phylogenetic analysis divided the 13 species into two primary clades, with the closest relationships found between I. batatas and Ipomoea trifida, as well as between Ipomoea platensis and I. digitata. Conclusions: These results enhance our understanding of the diversity among Ipomoea species and provide valuable insights for breeders using these species to generate improved varieties.

Detection of oomycete pathogens in UK peat-free growing media and implications for plant health

ABSTRACT Peat-based growing media products are being phased out in the UK. Alternative constituents may be at risk of harbouring oomycete plant pathogens. Thirty-five peat-free growing media samples were tested for oomycete pathogens by baiting and DNA metabarcoding. No Phytophthora species was detected by baiting, but seven species of Pythium, Phytopythium and Elongisporangium, including four species not previously reported in the UK, were baited. DNA matching eleven Phytophthora species was detected by metabarcoding across a range of samples. A coir sample was also found to contain DNA of Peronosclerospora, a genus of tropical downy mildew pathogens. DNA matching six Phytophthora pathogens, including P. bishii, a pathogen of strawberry not previously reported in the UK, was detected in recycled coir that had been used for strawberry production in The Netherlands. We discuss the implications of the species detected in terms of risks to plant health and the need for further evidence-gathering.

Does time matter? Intraspecific diversity of ribosomal RNA genes in lineages of the allopolyploid model grass Brachypodium hybridum with different evolutionary ages

BackgroundPolyploidisation often results in genome rearrangements that may involve changes in both the single-copy sequences and the repetitive genome fraction. In this study, we performed a comprehensive comparative analysis of repetitive DNA, with a particular focus on ribosomal DNA (rDNA), in Brachypodium hybridum (2n = 4x = 30, subgenome composition DDSS), an allotetraploid resulting from a natural cross between two diploid species that resemble the modern B. distachyon (2n = 10; DD) and B. stacei (2n = 20; SS). Taking advantage of the recurrent origin of B. hybridum, we investigated two genotypes, Bhyb26 and ABR113, differing markedly in their evolutionary age (1.4 and 0.14 Mya, respectively) and which resulted from opposite cross directions. To identify the origin of rDNA loci we employed cytogenetic and molecular methods (FISH, gCAPS and Southern hybridisation), phylogenetic and genomic approaches.ResultsUnlike the general maintenance of doubled gene dosage in B. hybridum, the rRNA genes showed a remarkable tendency towards diploidisation at both locus and unit levels. While the partial elimination of 35S rDNA units occurred in the younger ABR113 lineage, unidirectional elimination of the entire locus was observed in the older Bhyb26 lineage. Additionally, a novel 5S rDNA family was amplified in Bhyb26 replacing the parental units. The 35S and 5S rDNA units were preferentially eliminated from the S- and D-subgenome, respectively. Thus, in the more ancient B. hybridum lineage, Bhyb26, 5S and 35S rRNA genes are likely expressed from different subgenomes, highlighting the complexity of polyploid regulatory networks.ConclusionComparative analyses between two B. hybridum lineages of distinct evolutionary ages revealed that although the recent lineage ABR113 exhibited an additive pattern of rDNA loci distribution, the ancient lineage Bhyb26 demonstrated a pronounced tendency toward diploidisation manifested by the reduction in the number of both 35S and 5S loci. In conclusion, the age of the allopolyploid appears to be a decisive factor in rDNA turnover in B. hybridum.

Small sharks, big problems: DNA analysis of small fins reveals trade regulation gaps and burgeoning trade in juvenile sharks.

Many shark species have been overexploited for international markets, including fins for shark fin soup in Southeast Asia. Previous studies highlighted the value of large, threatened shark species, regulated under CITES Appendix II. However, sampling biases may have overlooked small shark species. Here, we address this by identifying species from ~4000 small shark fins in Hong Kong. These fins included species not recorded in previous surveys, raising the market's species diversity to 106. Nearly 75% of the small fins came from small shark species and 58.1% of small species were threatened with extinction. We identified an important CITES listing gap: Trade in 19 small, threatened species, especially from the family Triakidae, is unregulated. In addition, a quarter of small fins come from large sharks, indicating that substantial exploitation of juveniles is occurring and may be affecting fisheries sustainability. Enhanced surveillance of small shark fin trade is essential to ensure effective conservation under emerging trade regulations.

The clinical utility of plasma circulating tumor DNA in the diagnosis and disease surveillance in non-diffuse large B-cell non-Hodgkin lymphomas.

Aim: Advances in circulating tumor DNA (ctDNA) analysis for diffuse large B-cell lymphoma (DLBCL) have prompted the evaluation of its utility in other non-Hodgkin lymphomas (NHLs), leading to significant insights into its potential applications.Methods: We retrospectively studied paired plasma and tissue/bone marrow biopsies of 203 non-DLBCL NHLs [87 follicular lymphomas (FL), 64 mantle cell lymphomas (MCL), 30 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL) and 22 marginal zone lymphomas (MZL)]. Genomic profiling was performed using a targeted next generation sequencing panel (Hemasalus™). Longitudinal analyses were performed to explore plasma ctDNA utility in disease monitoring.Results: High plasma ctDNA detection rates were observed across NHL subtypes (FL: 88.5%, MCL: 90.6%, CLL/SLL: 100%, MZL: 68.2%), with high concordance of actionable mutations (FL: 87.4%, MCL: 93.8%, CLL/SLL: 93.3%, MZL: 81.8%) and multiple genetic aberrations exclusively identified in plasma. Particularly, IGH-BCL2 and IGH-CCND1 fusions were concordant between plasma and tumor biopsies in FLs (91.1%) and MCLs (91.3%), respectively. Longitudinal data demonstrated that ctDNA clearance correlated with complete response but ctDNA increases preceded radiological relapses.Conclusion: ctDNA exhibited high concordance with tumor biopsy in detecting genetic aberrations and demonstrated potential as a promising noninvasive approach to disease surveillance in non-DLBCL NHLs.

Identification of New Cultivar and Different Provenances of Dendrocalamus brandisii (Poaceae: Bambusoideae) Using Simple Sequence Repeats Developed from the Whole Genome.

Dendrocalamus brandisii is a high-quality bamboo species that can be used for both bamboo shoots and wood. The nutritional components and flavors of D. brandisii vary from different geographical provenances. However, the unique biological characteristics of bamboo make morphological classification methods unsuitable for distinguishing them. Although the new cultivar 'Manxie No.1' has significant differences in the branch characteristics and the color of shoot sheaths compared to the D. brandisii, it still lacks precise genetic information at the molecular level. This study identified 231,789 microsatellite markers based on the whole genome of D. brandisii and analyzed their type composition and distribution on chromosomes in detail. Then, using TP-M13-SSR fluorescence-labeling technology, 34 pairs of polymorphic primers were screened to identify the new cultivar 'Manxie No.1' and 11 different geographical provenances of D. brandisii. We also constructed DNA fingerprinting profiles for them. At the same time, we mapped six polymorphic SSRs to the gene of D. brandisii, among which SSR673 was mapped to DhB10G011540, which is related to plant immunity. The specific markers selected in this study can rapidly identify the provenances and the new cultivar of D. brandisii and help explore candidate genes related to some important traits.

Genetic fingerprinting distinguishes pathogenic variability of asiatic citrus canker pathogen, Xanthomonas citri pv. citri type A

Citrus canker, caused by Xanthomonas citri pv. citri (Xcc), is one of the most widespread citrus disease. In this study, 54 isolates of Xcc were obtained from citrus canker-infected leaves, twigs and fruit samples collected from various citrus-growing regions in Bangladesh. These isolates were analysed through pathogenicity tests and PCR amplification. The results revealed varying levels of aggressiveness among the isolates, ranging from weak to moderate and aggressive. No correlation was found between the host plants and their geographical locations. The genetic analysis of Xanthomonas isolates from around the world revealed 100 % similarity. All the Xanthomonas isolates were grouped into 2 major clusters. DNA fingerprinting showed high genetic variability, with no correlation between hosts, geographic locations or aggressiveness levels. This information on the population diversity of the citrus canker pathogen, Xanthomonas citri pv. citri (Xcc), will be valuable for developing sustainable management strategies for citrus cankers in Bangladesh and the broader Indian sub-continent region.

Examination of Opinions and Attitudes about Forensic DNA Database: Medical Students, Forensic Medicine Doctors and Clinical Geneticists

Examination of Opinions and Attitudes about Forensic DNA Database: Medical Students, Forensic Medicine Doctors and Clinical Geneticists

Development of a forensic DNA research grade test material.

Advancements in forensic DNA typing technology and methods have increased sensitivity and, while beneficial, carry the weight of more challenging profile interpretation. In response, the forensic DNA community has often requested more complex reference materials to address commonly encountered measurement and interpretation issues such as complex DNA mixtures, DNA degradation, and PCR inhibition. The National Institute of Standards and Technology (NIST) released Research Grade Test Material 10235: Forensic DNA Typing Resource Samples to support the forensic DNA community. Components include three single source samples, two degraded samples, and three mixture samples. As part of the Research Grade Test Material (RGTM) process, automated methods for bottling, alternative sample tube types, and the addition of carrier RNA for stabilizing low-quantity samples were investigated. Both internal and external testing demonstrate the stability of the material over time at 4°C through qPCR testing. In the development of a data portal, users have been allowed to anonymously upload results and compare their data with NIST and others. This report describes the preparation and stability of this material.

Bottleneck Effect Analysis in Indigenous Poonchi Chicken Located Near International Border of India and Pakistan

Background: Poonch is the remotest district of Jammu, India located near to line of control with Pakistan. Poonchi chicken is the first native poultry breed of Jammu region which is being characterized and registered with National Bureau of Animal Genetic Resources. Present study is the first study on genetic bottleneck assessment in Poonchi chicken population using recommended microsatellite markers. This study in Poonchi chicken would be of immense use in tracing evolutionary origin and formulating effective conservation strategies for genetic diversity within breeds. Methods: Blood samples were collected randomly from Poonch and Rajouri districts of Jammu for DNA extraction and analysis. Microsatellite markers selected for the analysis were ADL0268, MCW0206, MCW0081, ADL0278, MCW0069, MCW0248, MCW0111, MCW0222, MCW0016 and LEI0094. To evaluate the Poonchi chicken for mutation drift equilibrium, Bottleneck software was used. Infinite allele model (IAM), two phase model of mutation (TPM) and stepwise mutation model (SMM) tests were done. Mode shift test was also used as another method of qualitative test for allele frequency. Result: Total number of alleles observed were 72, with the total number alleles ranged from 6 to 9. The results of all three models (IAM, TPM, SMM) in Sign test depicted the heterozygosity was more in the expected number of loci in Poonchi chicken as 5.97 (P less than 0.05), 5.93 (P less than 0.05), 5.90 (P less than 0.05) respectively. Whereas for Wilcoxon rank test the IAM, TPM and SMM revealed significant values of (P less than 0.05) deviation indicating that all the loci deviate from mutation-drift equilibrium. Mode shift test graph obtained depicted a slight shift from normal L-shaped curve indicating recent bottleneck effect in Poonchi chicken. This might have resulted in the loss of number of effective alleles which suggests a need of planning and implementing an effective breeding policy in order to preserve and promote this native breed.

Histochemical Analysis of DNA and RNA Identified by Methyl Green - Pyronin ‘Y’ Method and Feulgen’s Reaction Staining in Liver of Channa Punctatus

The light microscopy was to identify cells, storage products, tissue deposits, and microbial pathogens. The Assessment of surrounding tissue with special stains may reveal aspects of interest for the tissue. We illustrate the expected staining characteristics of control Channa punctatus liver tissue with Methyl Green -Pyronin ‘Y’ and Feulgen’s Reaction stains. The aim of this study was to characterize the normal structure of the liver of Channa punctatus, a carnivorous freshwater fish found in Hasanparthy Lake, using gross anatomy, histology and histochemistry. Anatomically, the liver presents C-shaped and only two lobes smaller right and bigger left. The gallbladder is located in right lobe and shows elongated shaped. Histological analysis demonstrated that the hepatic parenchyma is made of two hepatocytes plates surrounded by sinusoids. The fish moreover pancreatic tissue was observed in visceral portion of liver, formed by exocrine pancreatic tissue and islet organ, constituting an extrahepatic pancreas. The nucleic acids content in different tissues in the freshwater fish of Channa punctatus. The fish were scarified and the tissues such as liver and Pancreas removed and processed for determination of nucleic acids. The results obtained in the present study may provide a contribution to the knowledge of the characteristics of nucleic acid as parameters of estimation of DNA and RNA levels in Liver of the fish.

Phage Display Selection of Antibody Libraries: Screening of Selected Binders.

Phage display selection of antibody libraries is a powerful method for generating and evolving monoclonal antibodies. The pComb3 phagemid family of phage display vectors facilitates the mining of antibody libraries in Fab format from human and nonhuman antibody repertoires. Here, we describe the screening for monoclonal Fab binders after selection of a polyclonal pool of Fab binders to an antigen of interest, with the goal of identifying and sequencing monoclonal antibodies that bind the antigen with high affinity and specificity. The screening cascade involves a phage ELISA, followed by a crude Fab ELISA and DNA fingerprinting and sequencing. The protocol outlines phage and crude Fab ELISAs using purified antigen immobilized on microplates, native antigen expressed on eukaryotic cells, or both.

First Report of Puccinia leontodontis on Scorzoneroides autumnalis from Iceland

Scorzoneroides autumnalis, commonly known as fall dandelion or autumn hawkbit, is a perennial plant with branched stems and several flower heads with bright yellow florets. In September 2023, rust disease was observed on 25% of plants present in two areas of Iceland showing reddish-yellow spots on the older leaves of the basal rosette. Morphological characteristics and sequencing analysis identified the rust species as Puccinia leontodontis. Our analyses confirm that P. leontodontis and P. hieracii are different species distinguishable through DNA analyses and by host genera, despite previously being regarded as synonyms. To our knowledge, this is the first report of P. leontodontis in Iceland.

Prevalence and Diversity of Plant Parasitic Nematodes in Irish Peatlands

The prevalence of plant parasitic nematodes (PPN) in the Irish peatlands was investigated in five different peatland habitats—raised bog, cutover scrub/woodlands, fens and peat grasslands, which were further sub-categorised into fourteen different sub-habitats. Within the raised bog habitat were healthy bog hummock (HBH), healthy bog lawn (HBL), degraded bog hummock (DBH) and degraded bog lawn (DBL) and the fen habitats were fen peat (FP) and rich fen peat (R-FP). Cutover scrub or woodland habitat included cutover scrub rewetted (C-RW), cutover scrub non-rewetted (C-NRW), woodlands rewetted (W-RW) and woodlands non-rewetted (W-NRW). Grassland included wasted peat (WP), rough grazing (RG-I) and improved fen peat grassland (IFPG-RW and IFPG-NRW). Soil samples from peatlands were all collected between July and December 2023 when the temperature ranged from 12 to 20 °C. One half of each sample was used for molecular nematode analysis and the other half for morphological identification of nematodes. For the morphological identification, a specific nematode extraction protocol was optimised for peatland soils, and the extracted nematodes were fixed onto slides to be studied under a high-power light microscope. Subsequently, the other part of the soil was processed to isolate total DNA, from which the 18S rRNA gene was sequenced for the identification of nematode taxa. The extracted DNA was also used for randomly amplified polymorphic DNA (RAPD) fingerprinting analysis to determine banding patterns that could classify different bog habitats based on PPN random primers. Compared to that in the climax habitats (HBH, HBL, DBH, DBL, FP, R-FP), PPN prevalence was recorded as being higher in grasslands (WP, RG-I, IFPG-RW and IFPG-NRW) and scrub/woodland ecosystems (C-RW, C-NRW, W-RW, W-NRW). The results indicate that nematode populations are different across the various bog habitats. Emerging and current quarantine PPN belonging to the families Pratylenchidae, Meloidogynidae, Anguinidae and Heteroderidae were noted to be above the threshold limits mentioned under EPPO guidelines, in grassland and wooded peatland habitats. Future actions for PPN management may need to be considered, along with the likelihood that these PPN might impact future paludiculture and other crops and trees growing in nearby agricultural lands.