DNA Analysis Research Articles

The Cretaceous diversification of angiosperms: perspectives from mesofossils

Abstract The plant fossil record during the Cretaceous documents a major transition in the dominant group of terrestrial autotrophs, as plant communities from the earlier Mesozoic were transformed by the appearance and rapid diversification of angiosperms. This transformation began in the Early Cretaceous, continued through the Late Cretaceous and led ultimately to the dominance of angiosperms in most terrestrial ecosystems today, which had profound consequences for the other organisms inhabiting terrestrial ecosystems and perhaps the planet as a whole. Our understanding of angiosperm diversification has been greatly improved over the past 50 years by integrated studies of fossil assemblages containing angiosperm pollen and leaves, but especially by new information from mesofossil floras that have provided previously unanticipated detail on floral form in Cretaceous angiosperms and have allowed the recognition of key dispersed pollen types in situ . Information from fossil flowers has greatly facilitated meaningful comparisons with living plants and integration with phylogenetic analyses of extant angiosperms based on DNA evidence. The combined insights from these discoveries provide a broadly consistent and coherent picture of angiosperm evolution through the Cretaceous, which comprises more than half of their entire evolutionary history.

Assessment of genetic homogeneity of in-vitro propagated apple root stock MM 104 using ISSR and SCoT primers

Apple is an important fruit crop that is always in demand due to its commercial and nutraceutical value. Also, the requirement for quality planting material for this fruit crop for new plantations is increasing continuously. In-vitro propagation is an alternative approach, which may help to produce genetically identical high grade planting material. In this study, for the first time, an efficient and reproducible propagation protocol has been established for apple root stock MM 104 via axillary bud. Culturing axillary buds on Murashige and Skoog apple rootstock (MM 104) resulted in better in-vitro propagation. (MS) basal medium supplemented with 3.0% (w/v) sucrose and 0.8% (w/v) agar. The axillary buds were established in MS basal medium with BA (5.0 µM), NAA (1.0 µM) and further used to establish invitro propagation protocol. Plant Growth Regulators (PGRs), BA (1.0 µM) in combination with NAA (1.0 µM) was found most efficient for shoot multiplication (100%) and produced 9.8 shoots/explants with an average shoot length of (2.4 ± cm). All the shoots produced roots in 0.1 µM IBA with a 5-day dark period. Acclimatization of in-vitro raised plantlets was obtained with vermiculite: perlite: sand: soil (2:2:1:1) resulting in 76% survival under field conditions. The study showed that the use of axillary bud is efficient for multiple-shoot production of apple rootstock (MM 104). This is the first comprehensive report on in-vitro growth of apple root stock MM 104 with an assessment of genetic stability using DNA fingerprinting profiles based on Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT). The genetic stability of in-vitro-produced plants, as determined by SCoT and ISSR primers, demonstrated genetic closeness to the mother plant.

An inter-laboratory comparison of probabilistic genotyping parameters and evaluation of performance on DNA mixtures from different laboratories

Probabilistic genotyping (PG) is becoming the preferred standard for evidence interpretation, amongst forensic DNA laboratories, especially those in the United States. Various groups have expressed concern about reliability of PG systems, especially for mixtures beyond two contributors. Studies involving interlaboratory testing of known mixtures have been identified as ways to evaluate the reliability of PG systems. Reliability means different things in different contexts. However, it suffices here to think about it as a mixture of precision and accuracy. We might also consider whether a system is prone to producing misleading results – for example large likelihood ratios (LRs) when the POI is truly not a contributor, or small LRs when the POI is a truly a contributor. In this paper we show that the PG system STRmix™ is relatively unaffected by differences in parameter settings. That is, a DNA mixture that is analyzed in different laboratories using STRmix™ will result in different LRs, but less than 0.05% of these LRs would result in a different, or misleading conclusion as long as the LR is greater than 50. For the purposes of this study, we define LRs assigned using different parameters for the same mixtures as similar if the LR of the true POI is greater than the LRs generated for 99.9% of the general population. These findings are based on an interlaboratory study involving eight laboratories that provided twenty known DNA mixtures of two to four contributors and their individual laboratory STRmix™ parameters. The eight sets of laboratory parameters included differences in STR kits and PCR cycles as well as the peak, stutter, and locus specific amplification efficiency variances.

4th International Conference on Forensic Sciences, Forensic Medicine and Criminology 2023 (INFOMEC 4.0)

The INFOMEC 2023 organized by the Management and Science University (MSU) is proud to celebrate the 4th anniversary of the International Conference on Forensic Science, Forensic Medicine, and Criminology. This is a premier gathering of experts from around the world who will be sharing their knowledge and expertise, exchange ideas, and discuss the latest advancements and techniques in the field. The conference features a diverse range of topics related to forensic science, forensic medicine, and criminology, including forensic pathology, forensic toxicology, forensic DNA analysis, crime scene investigation, forensic psychology, and criminal profiling, among others. Participants will have the opportunity to attend keynote presentations, poster sessions, and network with colleagues and peers from academia, government, and industry. The conference is an excellent platform for researchers, practitioners, and students to present their research findings and gain valuable feedback from experts in the field. Overall, the International Conference on Forensic Science, Forensic Medicine, and Criminology is an essential event for anyone involved in the fields of forensic science, forensic medicine, and criminology, providing an opportunity to stay up-to-date with the latest trends and developments, and contribute to the advancement of the field.

InDEL instability in two different tumoral tissues and its forensic significance.

There may be cases where malignant tumor samples can be used for forensic DNA profiling studies. STRs are the first systems preferred in forensic science laboratories for identification purposes. However, genetic instability in tumoral tissues causes STR polymorphism to change, leading to erroneous results. On the other hand, insertion/deletion polymorphism (InDels) are used as genetic markers in forensic science, as they have features that make both STR and SNPs preferable. Although previous studies approved that STR instability is observed in many different tumors, there are only a few studies that have displayed the instability of InDels in tumoral tissues before. In this study, it was aimed to determine whether instability is observed in formalin-fixed paraffin-embedded breast and thyroid tumoral tissues at 36plex InDel Panel. A total of 47 cases, 26 of which were diagnosed as breast cancer and 21 as thyroid cancer, were included in the study. In 21 of 26 (80.76%) breast cancers mutational changes were observed, however only 6 of 21 (28.57%) thyroid carcinoma cases displayed instability.Moreover, in these six cases, mutations were detected at only 1 or 2 loci. The most common change in both tissues was loss of heterozygosity. These findings suggest that paraffin embedded tissues of thyroid tumor can be used in cases of forensic genetic identification, however paraffin embedded breast cancer tissues should be examined with care. In conclusion, low InDel mutation rates compared to STR instability, make InDel analysis from paraffin blocks suitable for forensic genetic identification. However, researchers should keep in mind that there may be differences between the profiles of the tumoral tissues taken as reference and the actual case. In addition, by incorporating additional markers such as SNPs and microhaplotypes with low mutation rates into the study alongside Indels, researchers can significantly enhance the discrimination power in identification processes.

Identification and Comparison of DNA Fingerprinting of Mycobacterium Tuberculosis Isolates from Different Regions by IS 6110 RFLP Patterns

Identification and Comparison of DNA Fingerprinting of Mycobacterium Tuberculosis Isolates from Different Regions by IS 6110 RFLP Patterns

Establishing forensic DNA databases in Africa.

Establishing forensic DNA databases in Africa.

Modified differential lysis for sexual assault samples using a combined enzymatic and alkaline approach.

Traditional sexual assault sample processing methods are time-consuming and inefficient.This modified differential lysis method produces lysates with sufficient DNA yield and quality.A combined technique using enzymatic and alkaline lysis can accomplish fractional separation.Lysis with prepGEM and NaOH absent purification is compatible with downstream processes.

New records of seahorses (Teleostei: Syngnathidae: Hippocampus) from Kuwait in the Arabian (Persian) Gulf

A single species of seahorse, Hippocampus kuda, has been reported from the Arabian/Persian Gulf. Anatomical and DNA analyses of specimens from Kuwait indicate a second species occurs in the northern Gulf, and based on available specimens, may be the most prevalent species in the region. Evidence indicates the species to be H. trimaculatus and represents a range extension from India’s Gulf of Kutch of 2,600 km. DNA analysis of additional specimens is encouraged, particularly of H. trimaculatus specimens from outside the Gulf.

Authenticity and adulteration identification of Bubali Cornu based on DNA fingerprints

Whether adulteration exists is a difficult problem in the identification of traditional Chinese medicine(TCM). Bubali Cornu is mainly available in the medicinal material market in the form of buffalo horn silk or buffalo horn powder but lacks obvious identification characteristics, so there is a risk of adulteration. However, the method of identification of adulteration in Bubali Cornu is lacking at present. In order to ensure authenticity and identify adulteration of TCM Bubali Cornu, control the quality of TCM Bubali Cornu, and ensure the authenticity of clinical use, the DNA fingerprints of 43 batches of samples from pharmaceutical companies and medicinal material markets were identified, and the amplification primers of fluorescent DNA fingerprints of Bubali Cornu and Bovis Grunniens Cornu were screened. The DNA fingerprints of Bubali Cornu were obtained by fluorescent capillary typing. The identification effect of fluorescent capillary typing on different adulteration ratios was also tested. Two pairs of fluorescent STR typing primers, namely 16Sa and CRc, which can distinguish Bubali Cornu and Bovis Grunniens Cornu, were screened out, and a DNA fingerprint identification method was established. The 16Sa migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 223.4-223.9 bp and 225.5-226.1 bp. The CRc migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 518.8-524.8 bp and 535.9-542.5 bp. The peak height of the migration peak could be used for preliminary quantification of the adulterants with an adulteration ratio below 50%, and the quantitative results were similar to the adulteration ratio. In this study, a simple and quick universal DNA fingerprint method was established for the identification of Bubali Cornu and its adulterants, which could realize the identification of TCM Bubali Cornu and the semi-quantitative identification of the adulterants.

Analysis of DNA and RNA Genomic Profiles from Phase-II Clinical Trial NCT03398694 Showed Distinct Transcriptomic Profile for Patients With Local Failure

INTRODUCTION: Stereotactic radiosurgery (SRS) has become a standard of care for brain metastasis (BM). In the setting of lesions needing surgical intervention, it is typically delivered to the resection cavity after surgical resection to strike a balance between adverse effect from the dose of radiation and tumor recurrence. Pre-operative SRS is now being examined because of its ability to deliver radiation to all cells before surgical disruption and dispersion with added advantage of treating larger lesions with radiation. METHODS: We designed a phase II trial for patients with up to 4 BM and at least one resectable lesion. All lesions were treated with SRS and symptomatic lesions were resected. We examined both the clinical and biological outcomes. RESULTS: We performed whole genome DNA and RNA sequencing of the center and periphery of the resected lesion from the first 35 patients. We found that SRS causes significant and biologically observable damage to the tumor. Radiation induced DNA damage is qualitatively different between center and periphery of the tumor. Functionally distinct genetic profiles exist between both center and peripheral tumor locations as well as between different primary tumor types. Center of the tumor has higher functional mutation burden while peripheral samples initiate DNA repair pathways. Peripheral samples may interact more with surrounding environment when compared to central samples. Different radiation delivery modality has differential transcriptomic profile and patients who failed locally also had a distinct transcriptomic profile. CONCLUSIONS: In conclusion, radiation dosing fall-off has differential effect on the genomic landscape of the tumor and cells from the periphery are most likely gaining DNA repair abilities and that might explain tumor recurrence. Also, Gamma knife and LINAC as different genomic signature.

Microbiological quality of irrigation water on highly diverse fresh produce smallholder farms: elucidating environmental routes of contamination.

To evaluate the microbiological safety, potential multidrug-resistant bacterial presence and genetic relatedness (DNA fingerprints) of Escherichia coli isolated from the water-soil-plant nexus on highly diverse fresh produce smallholder farms. Irrigation water (n=44), soil (n=85), and fresh produce (n=95) samples from six smallholder farms with different production systems were analysed for hygiene indicator bacterial counts and the presence of shigatoxigenic E. coli and Salmonella spp. using standard microbiological methods. Identities of isolates were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the genetic relatedness of the E. coli isolates determined using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) analysis. Irrigation water E. coli levels ranged between 0 and 3.45 log MPN/100 ml-1 with five farms having acceptable levels according to the World Health Organization limit (3 log MPN/100 ml-1). Fresh produce samples on four farms (n=65) harboured E. coli at low levels (<1 log CFU/g-1) except for one sample from kale, spring onion, green pepper, onion, and two tomato samples, which exceeded international acceptable limits (100 CFU/g-1). Only one baby carrot fresh produce sample tested positive for Salmonella spp. Of the 224 samples, E. coli isolates were identified in 40% (n=90) of all water, soil, and fresh produce types after enrichment. Additionally, the DNA fingerprints of E. coli isolates from the water-soil-plant nexus of each respective farm clustered together at high similarity values (>90%), with all phenotypically characterized as multidrug-resistant. The clustering of E. coli isolated throughout the water-soil-plant nexus, implicated irrigation water in fresh produce contamination. Highlighting the importance of complying with irrigation water microbiological quality guidelines to limit the spread of potential foodborne pathogens throughout the fresh produce supply chain.

Detection of Barr bodies in exfoliated oral mucosal cells for sex determination using oral rinse-based cytology: A cross-sectional study

Background: Sex determination is essential in forensic science, anthropology, and medical diagnostics. Traditional methods, such as DNA analysis, are resource intensive and time-consuming. This pilot study explores the utility of oral rinse-based cytology for detecting Barr bodies in exfoliated oral mucosal cells from saliva as a more accessible and efficient alternative. Aim: This study aimed to assess the viability of rinse-based cytology in identifying sex through the detection of Barr bodies. Materials and Methods: In the study, 60 participants aged 20–30, free of lesions, and nonsmokers/nondrinkers, were evenly divided into male and female groups. Unstimulated saliva was collected from each participant between 9 and 10 am Participants were instructed to tilt their heads slightly forward to accumulate saliva and then spit into disposable sterile containers. After centrifugation, two smears were prepared and stained with Papanicolaou (PAP) and hematoxylin and eosin (H and E) stains. The smears were labeled (single-blinded) and examined for the presence of Barr body by two oral pathologists. Results: H and E stain had a mean Barr body count of 2.38 and a standard deviation of approximately 2.731, whereas PAP stain had a higher mean count of 3.57 with a standard deviation of 3.933. Levene’s test showed significant differences (P = 0.000) in variances between the staining methods (F = 21.001, df = 118, P &lt; 0.001), but the average Barr body count difference of about 1.183 with P = 0.058 was not statistically significant (P &gt; 0.05). Conclusion: Oral rinse-based cytology offers a noninvasive, cost-effective, and time-efficient approach to sex determination. While this pilot study demonstrates its potential, further research involving larger and more diverse populations is required to validate its reliability fully. This innovative approach has the potential to impact various scientific and forensic disciplines, offering a practical alternative to traditional methods.

Genetic analysis of Octopus cyanea reveals high gene flow in the South-West Indian Ocean.

Octopus cyanea (Gray, 1849), abundant in the South-West Indian Ocean (SWIO), constitutes a vital resource for both subsistence and commercial fisheries. However, despite this socioeconomic importance, and recent indications of overfishing, little is known about the population structure of O. cyanea in the region. To inform sustainable management strategies, this study assessed the spatio-temporal population structure and genetic variability of O. cyanea at 20 sites in the SWIO (Kenya, Tanzania, Mozambique, Madagascar, Mauritius, Rodrigues, and the Seychelle Islands) by complementary analysis of mitochondrial DNA (mtDNA) noncoding region (NCR) sequences and microsatellite markers. MtDNA analysis revealed a shallow phylogeny across the region, with demographic tests suggesting historic population fluctuations that could be linked to glacial cycles. Contrary to expectations, NCR variation was comparable to other mtDNA regions, indicating that the NCR is not a hypervariable region. Both nuclear and mtDNA marker types revealed a lack of genetic structure compatible with high gene flow throughout the region. As adults are sedentary, this gene flow likely reflects connectivity by paralarval dispersal. All samples reported heterozygote deficits, which, given the overall absence of structure, likely reflect ephemeral larval recruitment variability. Levels of mtDNA and nuclear variability were similar at all locations and congruent with those previously reported for harvested Octopodidae, implying resilience to genetic erosion by drift, providing current stock sizes are maintained. However, as O. cyanea stocks in the SWIO represent a single, highly connected population, fisheries may benefit from additional management measures, such as rotational closures aligned with paralarval ecology and spanning geopolitical boundaries.

Blood microbial signatures associated with mortality in patients with sepsis: A pilot study

Sepsis is a life-threatening illness caused by the dysregulated host response to infection. Nevertheless, our current knowledge of the microbial landscape in the blood of septic patients is still limited. Next-generation sequencing (NGS) is a sensitive method to quantitatively characterize microbiomes at various sites of the human body. In this study, we analyzed the blood microbial DNA of 22 adult patients with sepsis and 3 healthy subjects. The presence of non-human DNA was identified in both healthy and septic subjects. Septic patients had a markedly altered microbial DNA profile compared to healthy subjects over α- and β-diversity. Unexpectedly, the patients could be further divided into two subgroups (C1 and C2) based on β-diversity analysis. C1 patients showed much higher bacteria, viruses, fungi, and archaea abundance, and a higher level of α-diversity (Chao1, Observed and Shannon index) than both C2 patients and healthy subjects. The most striking difference was seen in the case of Streptomyces violaceusniger, Phenylobacterium sp. HYN0004, Caulobacter flavus, Streptomyces sp. 11-1-2, and Phenylobacterium zucineum, the abundance of which was the highest in the C1 group. Notably, C1 patients had a significantly poorer outcome than C2 patients. Moreover, by analyzing the patterns of microbe-microbe interactions in healthy and septic subjects, we revealed that C1 and C2 patients exhibited distinct co-occurrence and co-exclusion relationships. Together, our study uncovered two distinct microbial signatures in the blood of septic patients. Compositional and ecological analysis of blood microbial DNA may thus be useful in predicting mortality of septic patients.

(1097) - Utility of Combined Donor-Derived Cell-Free DNA and Peripheral Gene Expression Profiling in Heart Transplant Recipients

(1097) - Utility of Combined Donor-Derived Cell-Free DNA and Peripheral Gene Expression Profiling in Heart Transplant Recipients

Circulating microbiome DNA as biomarkers for early diagnosis and recurrence of lung cancer

Lung cancer mortality is exacerbated by late-stage diagnosis. Emerging evidence indicates the potential clinical significance of distinct microbial signatures as diagnostic and prognostic biomarkers across various cancers. However, circulating microbiome DNA (cmDNA) profiles are underexplored in lung cancer (LC). Here, whole-genome sequencing is performed on plasma of LC patients and healthy controls (HCs). Differentially enriched microbial species are identified between LC and HC. A diagnostic model is developed, which has a high sensitivity of 87.7% and achieves an AUC of 93.2% in the independent validation dataset. Crucially, this model demonstrates the capability to detect early-stage LC, achieving a sensitivity of 86.5% for stage I and 87.1% for tumors <1cm. In addition, we construct a cmDNA model for recurrence, which precisely predicts LC recurrence after surgery. Overall, this study highlights the significant alterations of cmDNA profiles in LC, indicating its potential as biomarkers for early diagnosis and recurrence.

Cell-free DNA assay for malignancy classification of high-risk lung nodules

ObjectiveAlthough low-dose computed tomography has been proven effective to reduce lung cancer–specific mortality, a considerable proportion of surgically resected high-risk lung nodules were still confirmed pathologically benign. There is an unmet need of a novel method for malignancy classification in lung nodules. MethodsWe recruited 307 patients with high-risk lung nodules who underwent curative surgery, and 247 and 60 cases were pathologically confirmed malignant and benign lung lesions, respectively. Plasma samples from each patient were collected before surgery and performed low-depth (5×) whole-genome sequencing. We extracted cell-free DNA characteristics and determined radiomic features. We built models to classify the malignancy using our data and further validated models with 2 independent lung nodule cohorts. ResultsOur models using one type of profile were able to distinguish lung cancer and benign lung nodules at an area under the curve metrics of 0.69 to 0.91 in the study cohort. Integrating all the 5 base models using cell-free DNA profiles, the cell-free DNA–based ensemble model achieved an area under the curve of 0.95 (95% CI, 0.92-0.97) in the study cohort and 0.98 (95% CI, 0.96-1.00) in the validation cohort. At a specificity of 95.0%, the sensitivity reached 80.0% in the study cohort. With the same threshold, the specificity and sensitivity had similar performances in both validation cohorts. Furthermore, the performance of area under the curve reached 0.97 in both the study and validation cohorts when considering the radiomic profile. ConclusionsThe cell-free DNA profiles-based method is an efficient noninvasive tool to distinguish malignancies and high-risk but pathologically benign lung nodules.

A subspecies of marbled newt (Triturus marmoratus) in the Iberian Peninsula newly resolved from congruent nuclear and mitochondrial dna data

Abstract The herpetofauna of the Iberian Peninsula is relatively well-researched, yet detailed studies, at least in part relying on molecular genetic data, continue to reveal taxa new to science, mostly species and subspecies. Newts of the genus Triturus are one such group with undiscovered yet taxonomically relevant variation, as shown by the recent description of new (sub)species of pygmy newts (T. pygmaeus, T. rudolfi). The marbled newt, Triturus marmoratus, shows an equally deep and geographically coherent spatial-genetic diversification. It is here shown that a northern and a southern group are characterized by different mitochondrial dna profiles and are also differentiated in morphometry and colouration pattern. With no firm evidence for selection against intermediate genotypes, the southern group is described at the subspecies level, as T. marmoratus harmannis ssp. nov. The subspecies’ contact zone is situated at ca. 41.5 northern latitude and stretches from the Atlantic coast near Porto, Portugal to the northeast of Madrid, Spain.

Directional Breeding Generates Distinct Genetic Diversity in Hybrid Turf Bermudagrass as Probed with Simple Sequence Repeat Markers

Bermudagrass (Cynodon spp.) is a drought-resistant warm-season turfgrass adapted to the southern and transitional zones in the United States. Multiple hybrid cultivars have been developed and released for use as turfgrass, and others are still undergoing development. Increasing genetic diversity of commercial cultivars is vital to stress tolerance. A DNA profiling study of 21 experimental selections from the Oklahoma State University turfgrass breeding program and 11 cultivars was conducted using 51 simple sequence repeat primer pairs across the bermudagrass genome. A pairwise genetic relationship analysis of the genotypes using 352 polymorphic bands showed genetic similarity coefficients ranging from 0.59 to 0.89. The average pairwise population differentiation values were 0.012 for the 11 cultivars and 0.169 for the 21 selections. A cluster analysis using the unweighted paired group with the arithmetic average method grouped the entries into six clusters. A correlation analysis identified different levels of pairwise genetic relationships among the entries that largely reflected parental relationship. Directional breeding and selection for cold hardiness or drought resistance created progeny that had distinct genetic diversity in the tested bermudagrasses. It is evident that an increase in genetic diversity of the existing cultivar pool with the release of one or more experimental selections for commercial use will strengthen and improve bermudagrass systems.