DNA Analysis Methods Research Articles

Private detection of relatives in forensic genomics using homomorphic encryption.

Forensic analysis heavily relies on DNA analysis techniques, notably autosomal Single Nucleotide Polymorphisms (SNPs), to expedite the identification of unknown suspects through genomic database searches. However, the uniqueness of an individual's genome sequence designates it as Personal Identifiable Information (PII), subjecting it to stringent privacy regulations that can impede data access and analysis, as well as restrict the parties allowed to handle the data. Homomorphic Encryption (HE) emerges as a promising solution, enabling the execution of complex functions on encrypted data without the need for decryption. HE not only permits the processing of PII as soon as it is collected and encrypted, such as at a crime scene, but also expands the potential for data processing by multiple entities and artificial intelligence services. This study introduces HE-based privacy-preserving methods for SNP DNA analysis, offering a means to compute kinship scores for a set of genome queries while meticulously preserving data privacy. We present three distinct approaches, including one unsupervised and two supervised methods, all of which demonstrated exceptional performance in the iDASH 2023 Track 1 competition. Our HE-based methods can rapidly predict 400 kinship scores from an encrypted database containing 2000 entries within seconds, capitalizing on advanced technologies like Intel AVX vector extensions, Intel HEXL, and Microsoft SEAL HE libraries. Crucially, all three methods achieve remarkable accuracy levels (ranging from 96% to 100%), as evaluated by the auROC score metric, while maintaining robust 128-bit security. These findings underscore the transformative potential of HE in both safeguarding genomic data privacy and streamlining precise DNA analysis. Results demonstrate that HE-based solutions can be computationally practical to protect genomic privacy during screening of candidate matches for further genealogy analysis in Forensic Genetic Genealogy (FGG).

Recent advancements in meat traceability, authenticity verification, and voluntary certification systems

The growing demand for transparency in the food industry has led to significant advancements in meat traceability. Ensuring the authenticity and origin of meat products is critical for consumer trust, public health, and compliance with regulations. This paper reviews recent innovations in meat traceability, with a focus on blockchain technology as a novel approach to ensuring traceability. Additionally, advanced methods for verifying meat authenticity and origin, such as isotope fingerprinting, DNA analysis, and spectroscopic methods, are discussed. The role of voluntary certification schemes in enhancing traceability and authenticity verification in the meat industry is also explored. The findings highlight the importance of integrating cutting-edge technologies and certification schemes to build a robust and transparent meat supply chain.

SpecGMM: Integrating Spectral Analysis and Gaussian Mixture Models for taxonomic classification and identification of discriminative DNA regions

Abstract Motivation Genomic Signal Processing, which transforms biomolecular sequences into discrete signals for spectral analysis, has provided valuable insights into DNA sequence, structure, and evolution. However, challenges persist with spectral representations of variable-length sequences for tasks like species classification and in interpreting these spectra to identify discriminative DNA regions. Results We introduce SpecGMM, a novel framework that integrates sliding window-based Spectral analysis with a Gaussian Mixture Model to transform variable-length DNA sequences into fixed-dimensional spectral representations for taxonomic classification. SpecGMM’s hyperparameters were selected using a dataset of plant sequences, and applied unchanged across diverse datasets, including mitochondrial DNA, viral and bacterial genome, and 16S rRNA sequences. Across these datasets, SpecGMM outperformed a baseline method, with 9.45% average and 35.55% maximum improvement in test accuracies for a Linear Discriminant classifier. Regarding interpretability, SpecGMM revealed discriminative hypervariable regions in 16S rRNA sequences–particularly V3/V4 for discriminating higher taxa and V2/V3 for lower taxa–corroborating their known classification relevance. SpecGMM’s spectrogram video analysis helped visualize species–specific DNA signatures. SpecGMM thus provides a robust and interpretable method for spectral DNA analysis, opening new avenues in genomic signal processing research. Availability and implementation SpecGMM’s source code is available at https://github.com/BIRDSgroup/SpecGMM.

Developmental validation of NeoTyper autosomal STR kit

BackgroundShort Tandem Repeats (STRs) are pivotal to efficient human DNA profiling. Including mini-STRs can further improve the assay's efficiency. The FBI's (Federal Bureau of Investigation) expanded Combined DNA Index System (CODIS) list contains twenty STRs for human identification. The NeoTyper Autosomal kit developed by Neom Scientific Solutions Pvt. Ltd. enables the amplification of 28 loci, which include markers from the expanded CODIS list, are National DNA Index System (NDIS) recommended markers, and comply with the NDIS recommended allelic range. In addition, the kit also includes 8 more markers including Penta D and Penta E. The NeoTyper Autosomal kit STR markers includes 14 mini-STR which makes it very effective for the human identification of the degraded samples like unidentified body remains.This is a developmental validation study for NeoTyper, based on the listed Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. MethodologyWe extracted the human genomic DNA from blood and FFPE (Formalin-Fixed Paraffin-Embedded) samples, and performed PCR amplification for the 28 loci using the developed kit, NeoTyper, which features a six-dye chemistry. The amplified products were run on 3500xL Genetic Analyzer. We used GeneMapper ID-X v.1.4 (Applied Biosystems) to analyse peak height and allele calling. Developmental validation parameters, including genetic characterization, analysis of sensitivity, female-male mixture, precision, and accuracy (repeatability and reproducibility), were investigated and analysed. Furthermore, intra- and inter-laboratory comparisons, as well as mini-STR analysis and validation were done. ResultsThe developed NeoTyper Autosomal kit demonstrated 100 % sensitivity up to a DNA concentration of 62.5 pg, as measured by the number of alleles called. The assay also demonstrated good precision (measured in terms of standard deviation, SD ranging between 0.60 and 0.77) and reproducibility, with alleles called at 100 % accuracy. Furthermore, precision was high (measured as SD ranging from 0.50 to 0.66), with a 100 % accuracy. In 90.9 % of the samples (10 out of 11 female-male sample varied ratio), the mixture analysis revealed 100 % of the alleles being called. A comparative analysis of mini-STRs in GlobalFiler and NeoTyper Autosomal kit revealed a 100 % accuracy of the NeoTyper mini-STRs. ConclusionIn conclusion, the NeoTyper Autosomal kit developed by Neom Scientific Solutions Pvt. Ltd. enables the amplification of 28 loci, which includes markers from expanded CODIS list; are NDIS accepted markers and comply with their recommended allelic range. The integration of mini-STRs in the kit demonstrates its efficacy and reliability, particularly when addressing the difficulties posed by degraded samples. This developmental validation study demonstrates consistent performance with high sensitivity, precision and accuracy (repeatability and reproducibility) which is indicative of an advanced and efficient system for human identification.

Developmental Validation of the Microreader 23HS Plex ID System: A Novel Supplementary Non-CODIS STR Multiplex Assay for Forensic Application.

A novel supplementary non-CODIS STR multiplex assay designated as the "Microreader 23HS Plex ID System" was developed. The Microreader 23HS Plex ID System enables simultaneous profiling of 23 STR loci and the amelogenin locus. The majority of these loci are non-CODIS STRs (D4S2408, D9S2157, D20S161, D3S2459, D18S1364, D13S305, D1S2142, D19S400, D6S1017, D7S1517, D2S1776, D2S1360, D3S1744, D16S3391, D3S1545, D11S4463, D20S85, D1S549, D10S2325, D21S2055), with the exception of three CODIS STRs (D2S441, D12S391, and D22S1045). Followed the recommendations of Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese validation standards, a comprehensive set of validation studies were conducted, encompassing PCR conditions, stutter ratio and peak height balance, sensitivity, precision and accuracy, reproducibility, species specificity, inhibition, as well as mixture testing. The results demonstrated that the Microreader 23HS Plex ID System is a reliable and robust assay, with well-balanced peak heights, high precision and accuracy, species specificity, and resistance to common inhibitors. The sensitivity of the assay was determined to be 0.125ng of template DNA. In mixture study, all minor alleles were detected in two-sample mixtures across various ratios (1:19, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 4:1, 9:1, and 19:1). In population study, a total of 500 unrelated individuals of Han ethnicity from East China were genotyped. The allele frequencies and forensic population genetic parameters were calculated, with a cumulative random match probability of 7.757×10-27, and a total power of discrimination exceeding 0.999,999,999,999,999,999,999,999,99. In conclusion, the Microreader 23HS Plex ID System shows promise as a valuable supplementary tool for forensic applications, particularly in addressing complex kinship testing and challenges posed by STR mutation.

Molecular diagnostics of familial hypercholesterolemia in Russia: yesterday, today and tomorrow

Familial hypercholesterolemia is a severe hereditary disease leading to the development of atherosclerosis and its complications in the form of angina pectoris, myocardial infarction, cerebral stroke, or even leading to sudden death. Since the description of the disease, the concept of it has undergone significant evolution. First, it became clear that the prevalence of this disease was significantly higher than originally thought (1:300 for heterozygous familial hypercholesterolemia and not as 1:500 as estimated earlier). Secondly, it has been established that it is not based on the pathology of the low-density lipoprotein receptor gene alone, but includes at least four monogenic forms (defects of the APOB, PCSK9, ARH genes) and may also have a multigenic nature. Thirdly, with the development of DNA analysis methods from the initially available Southern hybridization to next generation DNA sequencing, the exceptional molecular heterogeneity of familial hypercholesterolemia became obvious and, accordingly, the need to establish national spectra of mutations leading to the development of familial hypercholesterolemia was established. Researchers have moved from characterizing individual mutations to creating national registries and databases. Finally, research into the genetics of familial hypercholesterolemia has led to the emergence of new classes of cholesterol-lowering drugs. In Russia, molecular diagnostics of familial hypercholesterolemia has also undergone significant changes since the beginning of the study of familial hypercholesterolemia in 1987 and to the present, consideration of these changes formed the basis of this review.

A comprehensive evaluation of Cell Elution Method (CEM) for forensic DNA analysis from smoked cigarettes

Cigarette stubs are commonly encountered trace DNA samples at crime scenes. Standard laboratory practice typically involves direct lysis of the stub for DNA extraction, leading to the co-extraction of DNA-degrading and inhibiting constituents from smoke and tobacco. This process can result in lower-quality DNA profiles. There has been limited focus on developing specific sample processing techniques that minimize these degrading agents and inhibitors before DNA extraction, which could significantly enhance the quality of DNA profiles. This study evaluates a previously established Cell Elution Method (CEM) against the conventional Direct Lysis Method (DLM) for DNA extraction from cigarette stubs. DNA quantity, quality, and subsequent STR profiles were assessed in 80 smoked cigarette stubs, comprising both flavoured and unflavoured types. While CEM exhibited comparable DNA yield from both flavoured (0.17 ng) and unflavoured (0.19 ng) cigarettes, DLM showed significant variability in average DNA yield for unflavoured (0.05 ng) and for flavoured (0.25 ng) cigarettes. Notably, CEM-treated samples demonstrated lower Degradation Index (DI) values compared to DLM-treated ones for both the types of cigarettes. Consequently, STR profiling success rates were higher with CEM, with 95 % of flavoured and 55 % of unflavoured samples yielding informative profiles, compared to 80 % and 0 %, respectively, for DLM. In unflavoured stubs, Amelogenin marker amplification was achieved in 35 % of CEM-treated samples, significantly outperforming the 5 % success rate with DLM. Additionally, CEM resulted in higher average allele recovery rates for both flavoured (58.98 %) and unflavoured (33.41 %) samples compared to DLM. These findings indicate that CEM outperforms DLM in producing higher-quality DNA profiles from cigarette stubs. Thus, CEM can be a choice of method for processing cigarette stub prior to DNA extraction.

Shotgun DNA sequencing for human identification: Dynamic SNP selection and likelihood ratio calculations accounting for errors

Shotgun sequencing is a DNA analysis method that potentially determines the nucleotide sequence of every DNA fragment in a sample, unlike PCR-based genotyping methods that is widely used in forensic genetics and targets predefined short tandem repeats (STRs) or predefined single nucleotide polymorphisms (SNPs). Shotgun DNA sequencing is particularly useful for highly degraded low-quality DNA samples, such as ancient samples or those from crime scenes. Here, we developed a statistical model for human identification using shotgun sequencing data and developed formulas for calculating the evidential weight as a likelihood ratio (LR). The model uses a dynamic set of binary SNP loci and takes the error rate from shotgun sequencing into consideration in a probabilistic manner. To our knowledge, the method is the first to make this possible. Results from replicated shotgun sequencing of buccal swabs (high-quality samples) and hair samples (low-quality samples) were arranged in a genotype-call confusion matrix to estimate the calling error probability by maximum likelihood and Bayesian inference. Different genotype quality filters may be applied to account for genotyping errors. An error probability of zero resulted in the commonly used LR formula for the weight of evidence. Error probabilities above zero reduced the LR contribution of matching genotypes and increased the LR in the case of a mismatch between the genotypes of the trace and the person of interest. In the latter scenario, the LR increased from zero (occurring when the error probability was zero) to low positive values, which allow for the possibility that the mismatch may be due to genotyping errors. We developed an open-source R package, wgsLR, which implements the method, including estimation of the calling error probability and calculation of LR values. The R package includes all formulas used in this paper and the functionalities to generate the formulas.

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.

A Lateral Flow Biosensor Based on Isothermal Amplification for Visual Identification of Species and Sex from Bloodstain.

The prompt species identification from biological samples at a crime scene can rapidly filter out truly valuable biometric information for subsequent personal identification. Meanwhile, early sex determination can assist in narrowing the pool of suspects. However, the current methods for forensic DNA analysis, particularly in point-of-care scenarios, are often limited by the intricate equipment for signal generation and the laborious procedure for DNA purification. The present study introduces a novel portable lateral flow biosensor that possesses extraction-free and anti-aerosol characteristics for on-site determination of species and sex. The bloodstain can be directly submitted to loop-mediated isothermal amplification (LAMP) for the analysis of both mitochondrial and nuclear DNA. The incorporation of a lateral flow device with gold magnetic nanoparticle probes allows for visual interpretation of results through colorimetric signals while also preventing interference on result judgment from pigments such as hemoglobin. Carryover contamination, which is a disharmonious factor in LAMP, especially as the inherent contradiction derived from uncapping in the lateral flow strategy, has been effectively addressed through the integration of uracil DNA glycosylase without compromising the isothermy throughout the process. As a proof-of-concept experiment, species and sex can be accurately identified within 40 min from trace bloodstains amidst significant background interference by targeting cytochrome b and Y-chromosomal amelogenin. Furthermore, the single-blind study revealed a concordance rate of up to 100% in both simulative degraded and true dated bloodstains. This suggests that this biosensor has the potential to be utilized in forensic DNA analysis at crime scenes.

Early Non-Invasive Prenatal Testing at 6-9 Weeks of Gestation.

Non-invasive prenatal testing (NIPT) is usually performed beyond 10 weeks of gestation, because earlier in pregnancy, the fetal fraction is low, resulting in failure to obtain reliable results. This study aimed to evaluate the clinical performance of NIPT earlier in pregnancy using a method for cell-free DNA (cfDNA) analysis that eliminates the need for polymerase chain reaction (PCR), DNA sequencing, or microarrays (Vanadis® system, PerkinElmer, Waltham, MA, USA). Cell-free DNA was extracted from the maternal plasma of 30 singleton pregnancies at 6-9 weeks of gestation (group 1) and at 11-14 weeks of gestation of the same patients (group 2). The mean crown-rump length (CRL) and gestational age in group A was 16.12 mm and that in group B was 61.45 mm. In group A, results were obtained in all, but one, cases (97%). From the remaining pregnancies, one miscarried at 8 weeks and, therefore, the follow-up NIPT at 12 weeks could not be performed. The fetal sex was diagnosed correctly in the 28 cases that had a successful early test, and the results were in accordance with the examination at 12 weeks. There were no cases of aneuploidies and disomy was diagnosed correctly in all. The "Vanadis" prenatal NIPT assay can successfully be used early during the first trimester at 6-9 weeks of gestation (early NIPT) to identify the fetal sex. Further studies are needed to explore the diagnostic potential for aneuploidies.

Paying Tribute: Returning to the Story of the “Qur‘ān of ‘Uthmān

Objectives: The Qur’an occupies a central position in a religious and philosophical system that has had a significant and emblematic impact on human history, influencing Arabic studies worldwide. Such is particularly true for Russian Arabic studies, where the '‘Uthmanic Qur’an', with copies in Saint Petersburg and Uzbekistan, has played a crucial role. This study aims to honour our teachers and the Arab community of Qashqadarya, who continue to preserve their language and culture. Methodology: Synthetic, Comparative. Findings: The history of these manuscripts spans over twelve centuries, providing rich material linked to the fate of human lineages, nations, cities, peoples, and Islamic civilisation since its emergence in the Arabian Peninsula in the 7th century until the triumph of Islam. This Islam endured against the communist regime in the Islamic republics of the Soviet Union. The Russian School of Arabic Studies survived the 1917 Revolution due to its concentration on research areas that were of particular importance at the time. These included the study of Arabic sources on the history of the USSR territories and the living Arabic dialects used in the Central Asian republics. Such led to the publication of significant works on the language, ethnography, and history of the Arabs of Qashqadarya, whose ancestors brought the '‘Uthmanic Qur’an' from the Arabian Peninsula via the Silk Road to their land in Uzbekistan. Originality: Today, advanced research techniques must be used to study and analyse the '‘Uthmanic Qur’an' manuscript and reconstruct the origin, culture, and dialects of the Arab population in Qashqadarya, Uzbekistan. These include DNA analysis and modern linguistic methods based on big data and artificial intelligence technology.

O-117 Advances in PGT diagnostics - technologies beyond copy number

Abstract Genetic abnormalities are well recognised as the main factor contributing to the high rate of infertility, pregnancy loss and congenital abnormalities in humans. A prime example of this can be seen from recent studies showing that embryos identified as uniformly aneuploid following PGT-A have little to no reproductive potential. However, embryos shown to have no gross chromosomal abnormalities after PGT are not a guarantee of healthy pregnancy and many other undetected genetic issues are likely to influence embryo competence. Developments in both sample preparation and methods for DNA analysis are driving ever-increasing data yield from PGT platforms, permitting not only DNA quantitation, but also allowing qualitative evaluation of genotyping data in parallel. This opens the door to the possibility of overcoming some of the previous limitations of PGT, leading to a more comprehensive assessment of the genetic health and reproductive potential of embryos. How to use this increasing amount of information, and if it can tangibly improve test accuracy and clinical outcomes, remains open to debate. There is, for instance, no consensus on how to interrogate genotyping data for better distinction of clinically relevant aneuploidy as well as genome wide (ploidy) abnormalities and exogenous DNA contamination, which could impact the correct reporting of PGT results. There is also no consensus on the potential benefit of genotyping data to be used for the accurate distinction of meiotic recombination by linkage analysis, nor for fingerprinting of embryos to identify rare, but catastrophic, lab error events as a routine part of treatment. Finally, there is no clear path on how to utilise these improvements in embryo genotyping, to detect sub-chromosomal de novo genetic abnormalities that could impact embryo viability beyond the scope of current testing for chromosomal abnormalities alone. The aim of this presentation is therefore to focus on the current state of the ART and address the current and potential future uses of genotyping in PGT. Topics covered will include identification of biopsy sample contamination to determine the truth from the noise, through to how we can rescue embryos by utilising PGT as a molecular fertilisation check and how fingerprinting of embryo can be an invaluable tool in the clinic. The origin of aneuploidy will be explored and how genotyping is reshaping our understanding of aneuploidy landscape of the preimplantation embryo. From a more basic science perspective, the potential of genotyping to identify meiotic recombination will be discussed and how this fundamental element of human reproduction could be an important biomarker for reproductive competence. Lastly future perspectives on how the evolution of PGT technologies can bring us closer to the accurate detection of de novo genomic changes will be explored and how this is taking PGT-A beyond just aneuploidy detection. It is no secret that PGT for embryo selection continues to be a controversial area of reproductive medicine, and the field of PGT-A especially continues to show disquieting inconsistency in data analysis and reporting. This new era of genotyping brings with it information that can be used as a standardization tool helping to focus and facilitate improvements in the field of PGT for better and more consistent clinical reporting and outcomes.

DNA testing for investigative purposes: description of the perpetrator = Badania DNA dla celów dochodzeniowo-śledczych – opis sprawcy przestępstwa

The results of research projects to understand the diversity of the human genome have opened up new avenues of biomedical research and provided new tools for human identification studies. Genome wide association studies and epigenome wide association studies have enabled the identification of DNA markers that have been implemented and validated as predictive tools in the field of forensic DNA phenotyping. In the age of genomics, the study of biological traces can reveal the biogeographical ancestry, physical appearance, age and lifestyle of the perpetrator. The combination of different methods, including forensic genetic genealogy and prediction of phenotypic features, offers the possibility of significantly narrowing down the pool of suspects, thereby significantly improving the process of solving criminal cases. In general, a limitation is the availability of effective methods for large-scale DNA analysis that would ensure the forensic level sensitivity of the test.

Half-volume validation of the NGM Detect™ PCR Amplification Kit and its application on degraded casework samples.

The NGM Detect™ PCR Amplification Kit was designed particularly for genotyping degraded casework samples. This study aimed to validate the half-volume amplification of the kit and to present its successful long-term application. The validation was performed in accordance to the corresponding guidelines of the Scientific Working Group on DNA analysis methods and the European Network of Forensic Science Institutes. For validation parameters, such as sensitivity, reproducibility, and repeatability, polymerase chain reactions (PCR) were set up both manually and robotically, applying 29 cycles. For PCRs with sub-optimal DNA input (≤0.5 ng) the cycle numbers were increased to 31. Regardless of the PCR preparation method, the optimal 0.5 ng DNA input produced optimal allelic peak heights with no allelic dropout. The first alleles that failed to amplify started to appear at the level of 0.0375 ng input DNA, although the manually prepared PCRs produced fewer missing alleles. In this case, the raised cycle number produced 1.9% and 4.4% of dropout for manually and for robotically set up PCRs, respectively. In the case of 84 degraded casework samples, PCRs were prepared only by hand. The kit was able to provide informative profiles for 78.57%, 70.37%, and 69.77% for lowly, moderately, and highly degraded samples, respectively. Allelic dropouts were 26.05%, 44.88%, and 51.23% for the same groups. According to our results, we strongly recommend using the NGM Detect™ Kit in half-volume PCR system and encourage the usage of the kit in the particular cases when other kits fail to produce a complete DNA profile.

Exploring DNA Analysis Methods and Genetic Research Applications in Low and Middle-Income Nations: A Study of Sri Lanka

Using Sri Lanka as a case study, the paper explores how DNA analysis has transformed genetic research, particularly in low and middle-income countries (LMICs). It discusses various DNA analyzing techniques, from traditional methods like Sanger sequencing to advanced techniques such as next-generation sequencing (NGS) and CRISPR-Cas9, highlighting their applications in disease research, population genetics, and forensic science. Sri Lanka's advancements in genetic research, including DNA sequencing, typing, and recent developments in X-chromosome-based DNA typing, are emphasized. The paper also examines challenges and opportunities in LMICs regarding genetic research and underscores the importance of DNA analysis in advancing personalized medicine and understanding genetic diversity. Additionally, it discusses Sri Lanka's efforts in education and training in molecular biology. It explores the country's rich genetic diversity and demographic history, focusing on ethnic studies and historical interactions among different population groups. Overall, the paper highlights the significance of DNA analysis in genetic research and its potential implications for LMICs. Sri Lanka is a notable example of progress in the field.

Bioaerosol Sampling Devices and Pretreatment for Bacterial Characterization: Theoretical Differences and a Field Experience in a Wastewater Treatment Plant.

Studies on bioaerosol bacterial biodiversity have relevance in both ecological and health contexts, and molecular methods, such as 16S rRNA gene-based barcoded sequencing, provide efficient tools for the analysis of airborne bacterial communities. Standardized methods for sampling and analysis of bioaerosol DNA are lacking, thus hampering the comparison of results from studies implementing different devices and procedures. Three samplers that use gelatin filtration, swirling aerosol collection, and condensation growth tubes for collecting bioaerosol at an aeration tank of a wastewater treatment plant in Trieste (Italy) were used to determine the bacterial biodiversity. Wastewater samples were collected directly from the untreated sewage to obtain a true representation of the microbiological community present in the plant. Different samplers and collection media provide an indication of the different grades of biodiversity, with condensation growth tubes and DNA/RNA shieldTM capturing the richer bacterial genera. Overall, in terms of relative abundance, the air samples have a lower number of bacterial genera (64 OTUs) than the wastewater ones (75 OTUs). Using the metabarcoding approach to aerosol samples, we provide the first preliminary step toward the understanding of a significant diversity between different air sampling systems, enabling the scientific community to orient research towards the most informative sampling strategy.

Development and validation of a novel 30-plex STR assay for canine individual identification and parentage testing

Domestic dogs are helpers in outdoor human work and companions for families; thus, individual canine identification and parentage testing are crucial in certain fields, including forensics and breeding programs. In this study, a six-dye fluorescent labeling multiplex amplification system containing 29 canine short tandem repeats (STRs) and the sex-determining marker DAmel was developed. The system was called the Tronfo Canine 30-plex STR Kit and was further validated according to the Scientific Working Group on DNA Analysis Methods and the Organization of Scientific Area Committees for Wildlife Forensics guidelines, including tests for PCR conditions, precision, species specificity, sensitivity, stability, repeatability and reproducibility, a population study, and a study of 16 paternity test cases. The results indicated that the novel canine STR assay was accurate, specific, reproducible, stable, and robust. Complete profiles were obtained with 31.25 pg of canine DNA. Additionally, 500 unrelated canine individuals were investigated using this novel system, and the combined power of discrimination and exclusion values were 0.999999999999999999 and 0.999996451039850, respectively. These results suggest that the Tronfo Canine 30-plex STR Kit is highly polymorphic, informative, and suitable for individual canine identification and parentage testing.

Validation and forensic application of a new 36 X-STR loci multiplex system

Abstract X-chromosomal short tandem repeats (X-STRs) are indispensable in specific cases, distinct from autosomal and Y chromosome genetic markers. SureID® X37 is an innovative six-color fluorescence multiplex detection system that can simultaneously amplify 36 X-STR loci (including DXS6795, DXS7132, DXS8378, DXS10101, DXS10103, DXS10079, DXS10134, GATA172D05, HPRTB, DXS6810, DXS10135, DXS6797, DXS10074, DXS7424, DXS9902, DXS7423, DXS10148, DXS10162, DXS6809, GATA31E08, DXS6803, DXS10075, DXS6807, DXS10164, DXS6789, DXS10146, DXS981, DXS9895, DXS101, DXS6800 DXS907, and DXS8377) and the Amelogenin locus. In this study, we validated its suitability for forensic identification per SWGDAM (Scientific Working Group on DNA analysis methods) guidelines, including PCR conditions, precision, accuracy, case-type samples, sensitivity, repeatability, reproducibility, species specificity, stability, stutter calculation, and DNA mixtures. Additionally, 577 Han Chinese individuals were used to investigate the utility of the system in forensic population genetics. The results indicated that the system is sensitive, stable, and reliable, and is suitable for typical forensic case. Subsequent population investigations confirmed that it serves as a potent supplementary tool in forensic applications.

A DNA typing panel of 201 genetic markers for degraded samples: development and validation.

With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.