A DNA Methylation Signature from Buccal Swabs to Identify Tuberculosis Infection.

BACKGROUND: Tuberculosis (TB) is amongst the largest infectious causes of death worldwide and there is a need for a time- and resource-effective diagnostic method. In this novel and exploratory study, we show the potential of using buccal swabs to collect human DNA and investigate the DNA methylation (DNAm) signatures as a diagnostic tool for TB. METHODS: Buccal swabs were collected from pulmonary TB patients (n= 7), TB exposed (n= 7), and controls (n= 9) in Sweden. Using Illumina MethylationEPIC array the DNAm status was determined. RESULTS: We identified 5644 significant differentially methylated CpG sites between the patients and controls. Performing the analysis on a validation cohort of samples collected in Kenya and Peru (patients, n=26; exposed, n=9; control, n=10) confirmed the DNAm signature. We identified a TB consensus disease module, significantly enriched in TB-associated genes. Lastly, we used machine learning to identify a panel of seven CpG sites discriminative for TB and developed a TB classifier. In the validation cohort the classifier performed with an AUC of 0.94, sensitivity of 0.92, and specificity of 1. CONCLUSION: In summary, the result from this study shows clinical implications of using DNAm signatures from buccal swabs to explore new diagnostic strategies for TB.

Estimation of Human Chronological Age from Buccal Swab Samples through a DNA Methylation Analysis Approach of a Five-Locus Multiple Regression Model.

Recent advancements in forensic genetics have facilitated the extraction of additional characteristics from unidentified samples. This study delves into the predictive potential of a five-gene (ELOVL2, FHL2, KLF14, C1orf132, and TRIM59) methylation rate analysis for human age estimation using buccal swabs collected from 60 Italian volunteers. The methylation levels of specific CpG sites in the five genes were analyzed through bisulfite conversion, single-base extension, and capillary electrophoresis. A multivariate linear regression model was crafted on the training set, then the test set was employed to validate the predictive model. The multivariate predictive model revealed a mean absolute deviation of 3.49 years in the test set of our sample. While limitations include a modest sample size, the study provides valuable insights into the potential of buccal swab-based age prediction, aiding in criminal investigations where accurate age determination is crucial. Our results also highlight that it is necessary to investigate the effectiveness of predictive models specific to biological tissues and individual populations, since models already proven effective for other populations or different tissues did not show the same effectiveness in our study.

Postmortem age estimation via DNA methylation analysis in buccal swabs from corpses in different stages of decomposition-a "proof of principle" study.

Age estimation based on the analysis of DNA methylation patterns has become a focus of forensic research within the past few years. However, there is little data available regarding postmortem DNA methylation analysis yet, and literature mainly encompasses analysis of blood from corpses without any signs of decomposition. It is not entirely clear yet which other types of specimen are suitable for postmortem epigenetic age estimation, and if advanced decomposition may affect methylation patterns of CpG sites. In living persons, buccal swabs are an easily accessible source of DNA for epigenetic age estimation. In this work, the applicability of this approach (buccal swabs as source of DNA) under different postmortem conditions was tested. Methylation levels of PDE4C were investigated in buccal swab samples collected from 73 corpses (0-90 years old; mean: 51.2) in different stages of decomposition. Moreover, buccal swab samples from 142 living individuals (0-89 years old; mean 41.2) were analysed. As expected, methylation levels exhibited a high correlation with age in living individuals (training set: r2 = 0.87, validation set: r2 = 0.85). This was also the case in postmortem samples (r2 = 0.90), independent of the state of decomposition. Only in advanced putrified cases with extremely low DNA amounts, epigenetic age estimation was not possible. In conclusion, buccal swabs are a suitable and easy to collect source for DNA methylation analysis as long as sufficient amounts of DNA are present.

New targeted approaches for epigenetic age predictions.

BACKGROUND: Age-associated DNA methylation changes provide a promising biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. RESULTS: In this study, we utilized 4647 Illumina BeadChip profiles of blood to select CpG sites that facilitate reliable age-predictions based on pyrosequencing. We demonstrate that the precision of DNA methylation measurements can be further increased with droplet digital PCR (ddPCR). In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower correlation between chronological age and DNA methylation at individual CpGs, while the age-predictions were overall relatively accurate. Furthermore, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often associated with a CTCF binding site. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified, but reveals a stochastic pattern. Based on this, we have developed a new approach for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. CONCLUSION: Targeted DNA methylation analysis at few age-associated CpGs by pyrosequencing, BBA-seq, and particularly ddPCR enables high precision of epigenetic age-predictions. Furthermore, we demonstrate that the stochastic evolution of age-associated DNA methylation patterns in BBA-seq data enables epigenetic clocks for individual DNA strands.

Mitochondrial DNA methylation and copy number predict body composition in a young female population.

BACKGROUND: Since both genomic and environmental factors are involved in obesity etiology, several studies about the influence of adiposity on both nuclear DNA and mitochondrial DNA methylation patterns have been carried out. Nevertheless, few evidences exploring the usage of buccal swab samples to study mitochondrial DNA epigenetics can be found in literature. METHODS: In this study, mitochondrial DNA from buccal swabs collected from a young Caucasian population (n = 69) have been used to examine potential correlation between mitochondrial DNA copy number and methylation with body composition (BMI, WHtR and bioimpedance measurements). RESULTS: A negative correlation between mitochondrial DNA copy number and BMI was measured in females (p = 0.028), but not in males. The mean percentage of D-loop methylation is significantly higher in overweight than in lean female subjects (p = 0.003), and a specific CpG located in the D-loop shows per se an association with impaired body composition (p = 0.004). Body composition impairment is predicted by a combined variable including mtDNA copy number and the D-loop methylation (AUC = 0.785; p = 0.009). CONCLUSIONS: This study corroborates the hypothesis that mitochondrial DNA carries relevant information about body composition. However, wider investigations able to validate the usage of mtDNA methylation from buccal swabs as a biomarker are warranted.

Detection of DNA methylation from buccal swabs using nanopore sequencing to study stunting.

Stunting is the result of chronic malnutrition due to the lack of micronutrient-based methyl donors required for epigenetic programming during the first 1000 days of life. Methylation studies using bisulfite conversion from blood DNA are invasive and may not be practical for large-scale epidemiological investigation or nutrition intervention programs. Buccal epithelial methylation may reflect early germline methylation. Therefore, buccal cells can serve as convenient sample sources to collect biomarkers associated with the risk of stunting. This study aims to describe the feasibility of nanopore adaptive sampling in detecting DNA methylation from children's buccal DNA. We used adaptive sampling of Oxford Nanopore Technology on barcoded samples to describe differential methylation associated with malnutrition. Overall, the level of 5-methylcytosine (5mC) was lower in stunted children than in normal children. We also found differentially methylated regions at the MIR6724 and RNA45SN1 gene loci on chromosome 21, which was higher in stunted children than in normal children. We described and detected differential DNA methylation in the locus previously not known to be associated with stunting. Interestingly, this locus on chromosome 21 has been implicated in the stunted phenotype of Down syndrome.

Evaluation of direct PCR for routine DNA profiling of non-decomposed deceased individuals.

Forensic DNA profiling is a standard method used in the attempt to identify deceased individuals. In routine investigations, and if available, the preferred sample type is usually blood. However, this requires the invasive re-opening of the body, days or weeks after the autopsy, which is undesirable in resource-constrained mortuary settings. Motivated by the ease of sampling as well as reduced health and safety risks, this study aimed to establish the success rate of generating a full DNA profile on first attempt from buccal swab lysates using a direct PCR approach. Buccal swab samples were collected from 100 unidentified deceased males, and were subjected to direct DNA profiling with use of the Promega PowerPlex® Y23 Kit. At the time of sample collection, these individuals had been stored for between 1 and 887 days. This study shows that full DNA profiles were initially obtained from 73% of samples, which constitutes the first empirical data pertaining to first time success rates of direct PCR from post-mortem buccal lysates. Further investigation of partial and failed DNA profiles using real-time PCR showed that samples did not contain PCR inhibitors, DNA was not degraded, but DNA concentration was particularly low. Repeating DNA profiling with increased lysate input and extra PCR cycles yielded an additional six full DNA profiles, resulting in an overall success rate of 79%. Overall, DNA profile success rate was not associated with the duration of storage (p = 0.387). Lastly, massively parallel sequencing with the ForenSeq™ Signature DNA Prep kit provided more informative profiles for three additional samples. These results indicate that blood should therefore remain the sample of choice in a post-mortem setting, yet buccal lysates hold potential to be optimised further, which may ease the human identification workflow.

Assessment of the potential investigative value of a decentralised rapid DNA workflow for reference DNA samples.

Rapid DNA technology has the ability to provide DNA analysis results in real-time. The purpose of this study was to assess the potential investigative value that could be gained by using a rapid DNA workflow in Australia, for reference DNA samples. A survey of police charging stations and DNA analysis laboratories in five Australian jurisdictions identified that a rapid DNA analysis workflow could have impacted the decision to release a person of interest from custody in 0.6% cases. This equates to approximately 480 people per year across Australia who could be held in custody because of DNA results obtained in real time. The survey also identified the extent of a sample backlog in one jurisdiction and highlighted practices in place which limited the number of reference DNA samples collected, thereby reducing the potential impact of the DNA database. It is anticipated that while the cost of rapid DNA technology remains significantly greater than current laboratory processing, the uptake of the technology in Australia will be limited. However, as costs reduce, rapid DNA instruments could potentially become a viable option for the future of forensic science in Australia.

The expression of 10 candidate specific microRNA markers for human body fluid identification in animal buccal swabs.

MicroRNAs (miRNAs) have been of interest in forensic science for body fluid identification with recent years. However, there is no study investigating the species specificity of miRNA markers by the SYBR Green method. Due to the conservation of miRNAs across species, miRNA markers maybe less species-specific than mRNA markers, and in forensic cases, animal buccal swabs are more likely to appear. Therefore, in this study we addressed the influence of 8 kinds of animal buccal swabs on human saliva, semen, vaginal secretion swabs and blood identification with 10 candidate specific miRNA markers by the SYBR Green quantitative PCR. Our data showed that the expression levels of the candidate specific miRNA markers miR-124a and 372 in the cat, dog, mouse and rabbit buccal swabs were in the same range as the human vaginal secretion swabs; buccal swabs from these animals also showed similar expression levels to human saliva for the candidate specific miRNA markers miR-200c, 205 and 658. These results indicated that biomaterials of buccal swabs from cats, dogs, mice and rabbits may be mistaken for human saliva or human vaginal secretion swabs, both of which could result in false positives for human body fluids. Thus, the interpretation of these miRNA profiles for human body fluid identification can be inaccurate in the presence of these animal buccal swabs. Therefore, we suggested performing species tests before human body identification with miRNA markers.

Developmental validation of the ANDE™ rapid DNA system with FlexPlex™ assay for arrestee and reference buccal swab processing and database searching.

A developmental validation was performed to demonstrate reliability, reproducibility and robustness of the ANDE System with the FlexPlex assay, including an integrated Expert System, across a number of laboratories and buccal sample variations. Previously, the related DNAscan™/ANDE 4C Rapid DNA System using the PowerPlex®16 assay and integrated Expert System Software received NDIS approval in March 2016. The enhanced ANDE instrument, referred to as ANDE 6C, and the accompanying 6-dye, 27-locus STR assay, referred to as FlexPlex, have been developed to be compatible with all widely used global loci, including the expanded set of the CODIS core 20 loci. Six forensic and research laboratories participated in the FlexPlex Rapid DNA developmental validation experiments, testing a total of 2045 swabs, including those obtained from 1387 unique individuals. The goal of this extensive and comprehensive validation was to thoroughly evaluate and document the ANDE System and its internal Expert System to reliably genotype reference buccal swab samples in a manner compliant with the FBI's Quality Assurance Standards and the NDIS Operational Procedures. The ANDE System, including automated Expert System analysis, generated reproducible and concordant results for buccal swabs when testing various instruments at different laboratories by a number of different operators. When testing a number of non-human DNAs, including oral bacteria, the ANDE System and FlexPlex assay demonstrated limited cross-reactivity. Potential PCR inhibitors were evaluated as part of the validation and no inhibition was detected. Reproducible and concordant profiles were generated from buccal swab samples collected with a limit of detection appropriate for buccal swab collections from arrestees. The precision and resolution of the System met industry standards for detection of microvariants and single base resolution. The integrated Expert System appropriately demonstrated the ability to correctly pass or fail profiles for CODIS upload without human review. During this comprehensive developmental validation, the ANDE System successfully interpreted over 2000 samples tested with over 99.99% concordant alleles. The data package described herein led to the ANDE System with the FlexPlex assay receiving NDIS approval in June 2018.

Evaluation of buccal swabs for pharmacogenetics.

OBJECTIVE: A simple, non-invasive sample collection method is key for the integration of pharmacogenetics into clinical practice. The aim of this study was to gain samples for pharmacogenetic testing and evaluate the variation between dry-flocked and sponge-tipped buccal swabs in yield and quality of DNA isolated. RESULTS: Thirty-one participants collected samples using dry-flocked swabs and sponge-tipped swabs. Samples were assessed for DNA yield, quality and genotyping performance on a qPCR OpenArray platform of 28 pharmacogenetic SNPs and a CYP2D6 TaqMan copy number variant. DNA from sponge-tipped swabs had a significantly greater yield compared to DNA collected with dry-flocked swabs (p = 4.4 × 10-7). Moreover, highest genotyping call rates across all assays and highest CNV confidence scores were observed in DNA samples collected from sponge-tipped swabs (97% vs. 54% dry-flocked swabs; 0.99 vs. 0.88 dry-flocked swabs, respectively). Sample collection using sponge-tipped swabs provides a DNA source of sufficient quantity and quality for pharmacogenetic variant detection using qPCR.

Medical-grade buccal swabs versus drugstore cotton swabs: No difference in DNA yield.

We tested three types of medical-grade buccal swabs against standard cotton swabs for differences in DNA yield. A panel of swab types - one drugstore (Q-tips ® ) and three medical-grade - was used for buccal cell collection from three different individuals. DNA was extracted from all swabs using a QIAcube robot; quantitation values were measured by an Alu-based qPCR assay; and differences were compared through a 2-way ANOVA. Our results demonstrate that cotton swabs recover as much DNA as medical-grade swabs, but at a tremendously lower cost. Cotton swabs also display the greatest consistency of DNA yield, as indicated by the lowest coefficient of variation among the four tested swab types. These findings suggest that the use of standard cotton swabs for buccal cell collection offers not only a significant cost savings, but a more consistent method compared to the use of medical-grade swabs.

Developmental validation of the DNAscan™ Rapid DNA Analysis™ instrument and expert system for reference sample processing.

Since the implementation of forensic DNA typing in labs more than 20 years ago, the analysis procedures and data interpretation have always been conducted in a laboratory by highly trained and qualified scientific personnel. Rapid DNA technology has the potential to expand testing capabilities within forensic laboratories and to allow forensic STR analysis to be performed outside the physical boundaries of the traditional laboratory. The developmental validation of the DNAscan/ANDE Rapid DNA Analysis System was completed using a BioChipSet™ Cassette consumable designed for high DNA content samples, such as single source buccal swabs. A total of eight laboratories participated in the testing which totaled over 2300 swabs, and included nearly 1400 unique individuals. The goal of this extensive study was to obtain, document, analyze, and assess DNAscan and its internal Expert System to reliably genotype reference samples in a manner compliant with the FBI's Quality Assurance Standards (QAS) and the NDIS Operational Procedures. The DNAscan System provided high quality, concordant results for reference buccal swabs, including automated data analysis with an integrated Expert System. Seven external laboratories and NetBio, the developer of the technology, participated in the validation testing demonstrating the reproducibility and reliability of the system and its successful use in a variety of settings by numerous operators. The DNAscan System demonstrated limited cross reactivity with other species, was resilient in the presence of numerous inhibitors, and provided reproducible results for both buccal and purified DNA samples with sensitivity at a level appropriate for buccal swabs. The precision and resolution of the system met industry standards for detection of micro-variants and displayed single base resolution. PCR-based studies provided confidence that the system was robust and that the amplification reaction had been optimized to provide high quality results. The DNAscan integrated Expert System was examined as part of the Developmental Validation and successfully interpreted the over 2000 samples tested with over 99.998% concordant alleles. The system appropriately flagged samples for human review and failed both mixed samples and samples with insufficient genetic information. These results demonstrated the integrated Expert System makes correct allele calls without human intervention.

Evaluation of the RapidHIT™ 200, an automated human identification system for STR analysis of single source samples.

The RapidHIT™ 200 Human Identification System was evaluated to determine its suitability for STR analysis of single source buccal swabs. Overall, the RapidHIT™ 200 performed as well as our traditional capillary electrophoresis based method in producing useable profile information on a first-pass basis. General observations included 100% concordance with known profile information, consistent instrument performance after two weeks of buccal swab storage, and an absence of contamination in negative controls. When data analysis was performed by the instrument software, 95.3% of the 85 samples in the reproducibility study gave full profiles. Including the 81 full profiles, a total of 2682 alleles were correctly called by the instrument software, or 98.6% of 2720 possible alleles tested. Profile information was generated from as little as 10,000 nucleated cells, with swab collection technique being a major contributing factor to profile quality. The average peak-height-ratio for heterozygote profiles (81%) was comparable to conventional STR analysis, and while a high analytical threshold was required when offline profile analysis was performed (800 RFU), it was proportionally consistent with traditional methods. Stochastic sampling effects were evaluated, and a manageable approach to address limits of detection for homozygote profiles is provided. These results support consideration of the RapidHIT™ 200 as an acceptable alternative to conventional, laboratory based STR analysis for the testing of single source buccal samples, with review of profile information as a requirement until an expert software system is incorporated, and when proper developmental and internal validation studies have been completed.

Developmental validation of a fully integrated sample-to-profile rapid human identification system for processing single-source reference buccal samples.

UNLABELLED: Short tandem repeat (STR) DNA typing is a global standard for human identification. Current practice involves highly trained forensic analysts, operating in a laboratory setting, using multiple instruments to process samples and analyze the data. Here, we report the developmental validation of a fully integrated and automated DNA profiling system, the RapidHIT® System, capable of producing up to five high quality STR profiles with full controls in approximately 90min using PowerPlex®16 HS RapidHIT chemistry. The system integrates all sample handling steps: starting from lysis of cells on buccal swabs or other buccal sample types through DNA extraction, normalization, amplification,capillary array electrophoresis, detection, and integrated software analysis. The results describe the developmental validation of the RapidHIT™ System for buccal samples processed with the DNA IQ™ extraction chemistry using a guandinium chaotropic agent and paramagnetic beads followed by amplification using a modified version of PowerPlex 16 HS chemistry (PowerPlex 16 HS RapidHIT chemistry), and capillary electrophoresis with manual review of genotyping data following interpretation guidelines. All processing from the buccal swab to generation and processing of the profile occurs on the RapidHIT platform. RESULT: are concordant with traditional methods, with 88% first pass success rates for both the CODIS and PowerPlex 16 loci. Average peak height ratios were 0.89 for buccal swabs. The system produces full profiles from swabs with at least 176 ng of saliva DNA. Rapid DNA identification systems will significantly enhance capabilities for forensic labs, intelligence, defense, law enforcement, refugee and immigration applications, and kinship analysis.