Single Base Extension Research Articles

Phenotypic Classification of Eye Colour and Developmental Validation of the Irisplex System on Population Living in Malakand Division, Pakistan.

Eye colour digital photographs and buccal swab samples of 893 individuals of different age groups were collected. Multiplexed SNaPshot single base extension chemistry was used, and the genotypic results were analysed. Snapshot data were used for eye colour prediction through the IrisPlex and FROG-kb tool. The results of the present study found brown eye colour to be the most prevalent eye colour in comparison to intermediate and blue coloured. Overall, individuals with brown-coloured eyes possess CT (46.84%) and TT (53.16%) genotypes. Blue eye-coloured individuals are solely of the CC genotype, while individuals of intermediate eye colour carry CT (45.15%) and CC (53.85%) genotypes in rs12913832 SNP in the HERC2 gene. It was also revealed that brown-coloured eyes individuals were dominant among all age groups followed by intermediate and blue. Statistical analysis between particular variables and eye colour showed a significant p-value (<0.05) for rs16891982 SNP in SLC45A2 gene, rs12913832 SNP in HERC2 gene, rs1393350 SNP in SLC45A2, districts and gender. The rest of the SNPs were non-significant with eye colour, respectively. The rs12896399 SNP and SNP rs1800407 were found significant with rs16891982 SNP. The result also demonstrated that the study group differs from the world population based on eye colour. The two eye colour prediction results were compared, and it was discovered that IrisPlex and FROG-Kb had similar higher prediction ratios for Brown and Blue eye colour. The results of the current study revealed brown eye colour to be the most prevalent amongst members of the local population of Pakhtoon ethnicity in the Malakand Division of northern Pakistan. A set of contemporary human DNA samples with known phenotypes are used in this research to evaluate the custom panel's prediction accuracy. With the aid of this forensic test, DNA typing can be supplemented with details about the appearance of the person from whom the sample was taken in cases involving missing persons, ancient human remains, and trace samples. This study may be helpful for future population genetics and forensics studies.

Detection of 13 Hypervariable Region 1 (Hv1) SNPs using single-base extension (Sbe) primers in parallel with Sanger sequencing

The aim of this study was to determine whether single-base extension (SBE) chemistry can be applied to forensic practice of testing the target single nucleotide polymorphisms (SNPs) of the mitochondrial DNA (mtDNA) Hypervariable Region 1 (HV1). Despite itsweak discrimination power, high copy number of mtDNA per cell and its stability against degradation guarantee mtDNA testing a place in modern forensic genetics. In this research, buccal swab samples were obtained from 294 individuals from Bosnia and Herzegovina. Following DNA isolation using QIAamp® DNA Mini Kit, full sequencing of HV1 was completed using chain-termination Sanger sequencing method. SBE reactions were then performed by targeting 13 SNPs that were identified to be the most frequent in the study population. Uniplex SBE reactions for each individual SNP, as well as two multiplex reactions were prepared for both pure and mixed samples. The results showed complete agreement of the Sanger sequencing results with SBE reactions for both uniplex and multiplex reactions. The results obtained with SBE were encouraging in regard to multiplexing and processing of the mixed samples, since the allele of the minor contributor to the sample was observed in SBE electropherogram in all prepared mixtures. SBE method is limited by the fact that only target SNPs of interest will be analyzed, meaning that they must be carefully selected and curated for each population. However, typing with SBE protocol is accurate, as compared to the golden standard of Sanger sequencing, but was more time- and labor-efficient and simpler to analyze, therefore offering a suitable alternative when Sanger sequencing is not available, given that polymorphic SNPs are known for the population of interest.

Evaluation of DNA Methylation-Based Age-Prediction Models from Saliva and Buccal Swab Samples Using Pyrosequencing Data

In forensic genetics, the identification of an individual is often carried out by comparing unknown DNA profiles obtained in a case against databases or references. When no match is found, investigators need new tools in order to obtain additional leads. The latest technical advances now make it possible to predict externally visible characteristics. With this objective, predicting the age of an individual through DNA methylation analysis remains one of the last challenges. The prediction models have to account for the specific constraints of this field, including tissue specificity and DNA availability (i.e., low DNA amounts or low-quality DNA). Jung and colleagues have recently produced models from blood, saliva and buccal cells by using a single base extension sequencing method. With the goal of evaluating these models in our own analytical conditions, saliva and buccal cell samples from 115 French individuals between the ages of 0 and 88 years old were collected and analyzed. After having determined the optimal analysis conditions, including the DNA quantity for bisulfite conversion (75 ng), some differences were highlighted in the measured methylation rates between the two studies. Despite these discrepancies, the prediction performance levels remain very similar, our study showing mean absolute errors of 3.5 years, 3.9 years and 3.2 years, respectively, for the saliva, buccal swab and multitissue model, with limitations observed for the oldest and youngest individuals. Furthermore, we propose the use of a prediction interval with an error dispersion and correct prediction rate at ±5 years and ±10 years, respectively.

Forensic Age Estimation through a DNA Methylation-Based Age Prediction Model in the Italian Population: A Pilot Study

DNA methylation is one of the epigenetic marks which has been studied intensively in recent years for age predicting purposes in the forensic area. In order to integrate age prediction into routine forensic workflow, the purpose of this study was to standardize and optimize a DNA methylation-based protocol tailored to the Italian context. A previously published protocol and age-predictive method was implemented for the analysis of 84 blood samples originating from Central Italy. The study here presented is based on the Single Base Extension method, considering five genes: ELOVL2, FHL2, KLF14, C1orf132, now identified as MIR29B2C, and TRIM59. The precise and specific steps consist of DNA extraction and quantification, bisulfite conversion, amplification of converted DNA, first purification, single base extension, second purification, capillary electrophoresis, and analysis of the results to train and test the tool. The prediction error obtained, expressed as mean absolute deviation, showed a value of 3.12 years in the training set and 3.01 years in the test set. Given that population-based differences in DNA methylation patterns have been previously reported in the literature, it would be useful to further improve the study implementing additional samples representative of the entire Italian population.

Target-allele-specific probe single-base extension (TASP-SBE): a novel MALDI-TOF-MS strategy for multi-variants analysis and its application in simultaneous detection of α-/β-thalassemia mutations.

Single-nucleotide variants (SNVs) and copy number variations (CNVs) are the most common genomic variations that cause phenotypic diversity and genetic disorders. MALDI-TOF-MS is a rapid and cost-effective technique for multi-variant genotyping, but it is challenging to efficiently detect CNVs and clustered SNVs, especially to simultaneously detect CNVs and SNVs in one reaction. Herein, a novel strategy termed Target-Allele-Specific Probe Single-Base Extension (TASP-SBE) was devised to efficiently detect CNVs and clustered SNVs with MALDI-TOF-MS. By comprehensive use of traditional SBE and TASP-SBE strategies, a MALDI-TOF-MS assay was also developed to simultaneously detect 28 α-/β-thalassemia mutations in a single reaction system, including 4 α-thalassemia deletions, 3 HBA and 21 HBB SNVs. The results showed that all 28 mutations were sensitively identified, and the CNVs of HBA/HBB genes were also accurately analyzed based on the ratio of peak height (RPH) between the target allele and reference gene. The double-blind evaluation results of 989 thalassemia carrier samples showed a 100% concordance of this assay with other methods. In conclusion,a one-tube MALDI-TOF-MS assay was developed to simultaneously genotype 28 thalassemia mutations. This novel TASP-SBE was also verified a practicable strategy for the detection of CNVs and clustered SNVs, providing a feasible approach for multi-variants analysis with MALDI-TOF-MS technique.

An innovative single-base extension method for synchronous detection of point mutations and MSI status in colorectal cancer.

An accurate genotyping analysis is one of the critical prerequisites for patients with colorectal cancer receiving matched therapies. Conventional genotyping analysis is currently used to detect either gene mutations or MSI status, delaying the detection of critical tumor biomarkers and thus the optimal time for treatment. An assay that analyzes both biomarkers in a streamlined process is eagerly needed. We developed an assay combining Multiplex PCR Amplification, Single-base Extension and capillary electrophoresis (CE) analysis (MASE-CE) for synchronous detection of KRAS/NRAS/BRAF mutations and MSI status. In a 190 colorectal cancer cohort, we identified seven somatic mutations in KRAS, NRAS and BRAF as well as five MSI loci (D2S123/D5S346/D17S250/BAT-25/BAT-26) simultaneously. KRAS/NRAS/BRAF mutations were detected by NGS and MASE-CE, and MSI status were detected by PCR-CE and MASE-CE methods. The MASE-CE method showed high consistency with NGS for mutation detection (Kappa value ≥0.8) and PCR-CE (Kappa value=0.79). In addition, the limits of detection (LOD) of MASE-CE assay for MSI and somatic mutation were 5% and 2%, respectively. In somatic mutation detection and MSI detection, the LOD of MASE-CE assay was superior to that of qPCR and NGS. MASE-CE assay is a highly sensitive, time-saving and specimen-saving method, which can greatly avoid the cumbersome testing process and provide clinical decision for doctors in time.

Can we integrate method-specific age-predictive models?: Analysis method-induced differences in detected DNA methylation status

Forensic research surrounding the use of DNA methylation (DNAm) markers to predict age suggests that accurate prediction of chronological age can be achieved with just several DNAm markers. Several age-prediction models are based on DNAm levels that are detectable by a diverse range of DNAm analysis methods. Among the many DNAm analysis methods, targeted amplicon-based massively parallel sequencing (MPS) and single-base extension (SBE) methods have been widely studied owing to their practicality, including their multiplex capabilities. Since these two DNAm analysis methods share an identical amplification step during their experimental processes, several studies have compared the differences between the methods to construct integrated age-prediction models based on both MPS and SBE data. In this study, we compared the specific differences in DNAm levels between these two commonly exploited analysis methods by analyzing the identical PCR amplicons from the same samples and quantifying the actual bisulfite-converted DNA amount involved in the PCR step. The DNAm levels of five well-studied age-associated markers—CpGs on the ELOVL2, FHL2, KLF14, MIR29B2CHG, and TRIM59 genes—were obtained from blood samples of 250 Koreans using both DNAm analysis methods. The results showed that only ELOVL2 is interchangeable between the MPS and SBE methods, while the rest of the markers showed significant differences in DNAm values. These differences may result in high errors and consequential lowered accuracy in age estimates. Therefore, a DNAm analysis method-specific approach that considers method-induced DNAm differences is recommended to improve the overall accuracy and reliability of age-prediction methods.

Age prediction by methylation analysis of small amounts of DNA using locked nucleic acids.

Age prediction based on methylation analysis has been reported in many populations, with 10ng or more of DNA usually required for each determination. In this study, we designed thermostable locked nucleic acid (LNA) primers by replacing a small number of DNA bases in standard DNA primers with LNAs. We evaluated these primer sets by single-base extension analysis using 10, 5, or 2ng of DNA that would be less than template DNA used in standard methylation testing, and determined sensitivity and accuracy. We analyzed EDARADD, SST, and KLF14 genes, targeting one CpG site in each gene. Melting temperature values of most LNA primers were 4°C higher than those of DNA primers. The intensities of signals from the EDARADD and SST genes were significantly improved by the LNA primers, by 3.3 times and 1.4 times, respectively, compared with the DNA primers using 2ng of DNA. Coefficient of variation (CV) analysis was used to assess the accuracy of the determined methylation levels. CVs were increased using small amounts of DNA, but lower CVs were detected using LNA primers. We also showed high accuracy of age prediction for 51 individuals using LNA primers. The lowest mean absolute deviation was obtained using 10ng of DNA and was 3.88 years with the LNA primers. Thermostable PCR primers were simply designed, and the LNAs improved the sensitivity and accuracy of methylation analysis for 10ng or less of DNA.

DNA methylation analysis for smoking status prediction in the Chinese population based on the methylation-sensitive single-nucleotide primer extension method

In some criminal cases, the identity of suspect is unknown and there is no matching DNA profile in the DNA database. Forensic DNA Phenotyping can provide useful investigative information for these cases. Most forensic studies focus on visible characteristics rather than behavioral characteristics. However, smoking is prevalent in the Chinese population, and DNA methylation is the most promising biomarker for smoking. We collected 204 whole blood samples from the Chinese population and measured methylation levels of 9 smoking-related CpG loci using the methylation-sensitive single-nucleotide primer extension method (Ms-SnuPE). But the single-base extension primers of loci cg12803068 and cg21566642 contained other CpG sites, which may introduce bias, and only the other 7 CpG loci were included in subsequent statistical analysis. The methylation level of locus cg05575921 near the aromatic hydrocarbon receptor repressor (AHRR) gene was much lower in the current smoker group than in the never smoker group. To evaluate the ability of each of 7 CpG loci to predict smoking status, the logistic regression (LR) models were established separately, and locus cg05575921 had the best ability to predict smoking status compared with the other 6 loci. Then, combined (including loci cg19572487, cg05575921, cg23480021, cg23576855, cg21161138, cg01940273, and cg09935388) and stepwise (including loci cg05575921 and cg01940273) multinomial logistic regression (MLR) models were also established. Both combined and stepwise MLR models had good efficiencies in predicting smoking status, and outperformed the above 7 LR models. However, the accuracy, specificity and area under the curve (AUC) of stepwise MLR model in the testing dataset were slightly higher than those of combined MLR model, and the stepwise MLR model required less loci information. Therefore, the stepwise MLR model based on 2 significant CpG loci was more recommended model for predicting smoking status in the Chinese population, and the formula was as follow: P = 1/(1 +e-(10.621–10.005*cg05575921–8.770*cg01940273)). Mainly 2 CpG loci (cg05575921 and cg01940273) played a major role in the prediction of smoking status, and the other 5 CpG loci contributed less. Moreover, for evaluating the ability of each of 7 CpG loci to predict cigarette consumption, the polynomial regression formulas were established separately. As the adjusted R2 was between 0.00 and 0.20, the methylation levels of these 7 loci were not closely associated with the cigarette consumption. Our methylation assay is simple, economical, and available in conventional forensic laboratories, and may be useful in assessing the smoking status of unknown suspects.

MtDNA haplogroup A enhances the effect of obesity on the risk of knee OA in a Mexican population

To evaluate the influence of mitochondrial DNA haplogroups on the risk of knee OA in terms of their interaction with obesity, in a population from Mexico. Samples were obtained from (n = 353) knee OA patients (KL grade ≥ I) and (n = 364) healthy controls (KL grade = 0) from Mexico city and Torreon (Mexico). Both Caucasian and Amerindian mtDNA haplogroups were assigned by single base extension assay. A set of clinical and demographic variables, including obesity status, were considered to perform appropriate statistical approaches, including chi-square contingency tables, regression models and interaction analyses. To ensure the robustness of the predictive model, a statistical cross-validation strategy of B = 1000 iterations was used. All the analyses were performed using boot, GmAMisc and epiR package from R software v4.0.2 and SPSS software v24. The frequency distribution of the mtDNA haplogroups between OA patients and healthy controls for obese and non-obese groups showed the haplogroup A as significantly over-represented in knee OA patients within the obese group (OR 2.23; 95% CI 1.22–4.05; p-value = 0.008). The subsequent logistic regression analysis, including as covariate the interaction between obesity and mtDNA haplogroup A, supported the significant association of this interaction (OR 2.57; 95% CI 1.24–5.32; p-value = 0.011). The statistical cross-validation strategy confirmed the robustness of the regression model. The data presented here indicate a link between obesity in knee OA patients and mtDNA haplogroup A.

Optimizing Multiplex PCR for a Set of Malay Ancestry Informative Marker-Single Nucleotide Polymorphisms (AIM-SNPs) and Preliminary Analysis of Genotypes Between Malay and non-Malay Population.

Background: Inference of genetic ancestry is of great interest in many fields and one of the markers in these analyses is ancestry informative marker single nucleotide polymorphisms (AIMSNPs). The Malay population is an ethnic group located mainly in South East Asia and comprises the largest ethnicity in Malaysia.&#x0D; Objectives: To determine Malay ancestry, Yahya et al, 2017 selected 37,487 SNPs from the genotyping data collected by the Malaysian Node of the Human Variome Project and Singapore Genome Variation Project and referenced them against the data from the International HapMap Project Phase 3. The SNPs determined to be informative for ancestry were compiled into AIM-SNP panels, and from these a few SNPs were selected for optimization in preparation for single base extension reaction multiplexing.&#x0D; Methodology: The chosen AIMSNPs were optimized and validated on Malay and non-Malay populations. Genotyping was carried out on participants of self-reported Malay and non-Malay ancestry respectively and the data were compared for Malay and non-Malay population to investigate for significant differences in the genotype between Malay and non-Malay participants.&#x0D; Findings: The results showed great similarities between the Malay and non-Malay population, which may arise from many factors, and further optimization of more SNPs and genotyping is required to definitively conclude the validity of the AIM-SNP panels for Malay population&#x0D; Conclusion: Knowledge of ancestry is important to minimise spurious association. This pilot study gives a brief account of the optimization process and offers an insight into how this may be done in South East Asian populations.

Range-wide assessment of a SNP panel for individualization and geolocalization of bigleaf maple (Acer macrophyllum Pursh)

Illegal logging is a worldwide problem that degrades ecosystems, and even low-risk markets like the United States report ~1000 significant hardwood timber theft cases per year. Due to its high value, bigleaf maple (Acer macrophyllum Pursh) is a common target of timber theft in western North America. Using samples from Washington, USA, a single nucleotide polymorphism [SNP] assay was recently developed to facilitate timber poaching investigations in this species based on the single base extension Agena MassARRAY® genotyping system and 133 loci. Our study expands earlier evaluations to include the full 2000 km latitudinal range of bigleaf maple, using 1142 samples collected from California, USA to British Columbia, CAN. Populations in this study are equivalent to level 3 ecoregions, with sample sizes ranging from 28 (California Coast) to 191 (Washington Coast). Wood-derived DNA concentrations between 0.24 and 6 ng/μL yielded SNP call rates >92%, and DNA concentrations within this range did not influence genotyping call rates. DNA from different tissues showed a weak but significant difference in call rate, with DNA from wood showing a slightly higher call rate than leaves (99.4% vs 97.9%, respectively). Analysis of 108 sample replicates showed that missing genotypes occurred at 0.165% of all loci, and that allelic drop-outs and drop-ins each occurred at frequencies of 0.022% and 0.032%, respectively. Population-specific genotype profile probabilities for Pacific Northwest ecoregions were lower than 1.9×10−33; profile probabilities are higher (less powerful) in Californian trees due to lower variability across these SNP loci. Conversely, the geographic origin of trees from Southern Californian ecoregions could be predicted with lower error (8.5–11.7%) than trees from the Pacific Northwest (36–80%), based on random forest classification. This study provides a rich database for forensic individualization of bigleaf maple across its complete range in western North America.

Qualitative Single Base Extension assay for Identification of Single Nucleotide Polymorphism in Mumps Virus Genome

Objective: Objective of the study was to standardize and evaluate suitability of Single Base Extension (SBE) singleplex assay for rapid routine monitoring of genetic homogeneity of virus samples, from productivity improvement experiments in Mumps vaccine. A Single Base Extension assay is a simple, cost effective tool for quick identification of single nucleotide polymorphism (SNP) at the three known sites in Mumps vaccine virus genome. The assay can be developed as a singleplex or multiplex version with potential for detection of up to ten SNP bases in a single reaction.Methods: SNaPshot kit from Applied Biosystems (ThermoFisher) was used to identify the nucleotide bases at the three SNP sites in Mumps virus genome. Approximate sizing locations of individual primers were determined using the SNaPshot Primer Focus Kit, also from Applied Biosystems. Genetic analyzer 3500 was used for the capillary electrophoresis of the processed samples. Results obtained from the data collection software were analyzed using GeneMapper software. Findings: Nucleotide G was identified at locations 1073 and 11345 and nucleotide C was identified at location 5261 for all tested mumps vaccine samples, thus demonstrating consistency in genetic homogeneity at the three known SNP hotspots in experimental mumps vaccine virus samples. Conclusion: The singleplex SBE assay was found to be a reliable and relatively inexpensive tool for routine monitoring of genetic homogeneity of vaccine virus samples, with potential for multiplexing that would reduce the cost and assay time even further.

A Rapid, Visible, and Highly Sensitive Method for Recognizing and Distinguishing Invasive Fungal Infections via CCP-FRET Technology.

Invasive fungal infection (IFI) is one of the leading causes of death in the intensive care unit (ICU) due to its high morbidity and mortality among immunocompromised patients. Early diagnosis of IFI is typically infeasible because of the lack of clinical signs and symptoms. By virtue of the cationic conjugated polymer-based fluorescence resonance energy transfer (CCP-FRET) technology, we develop a rapid, visible, simple, and sensitive method for simultaneous detection and discrimination of three types of pathogens, including Candida albicans (C.albicans), Klebsiella pneumoniae (K.pneumoniae), and Cryptococcus neoformans (C.neoformans). The CCP-FRET system contains a CCP fluorescent probe and pathogen-specific DNA labeled with fluorescent dyes. These two components spontaneously self-assemble into the complex under electrostatic attraction, resulting in an efficient FRET from CCP to fluorescent dyes when irradiated with a 380 nm ultraviolet (UV) light. The CCP-FRET method can specifically identify the DNA molecules that are extracted from culture pathogen strains or blood samples via PCR and single base extension (SBE) reactions, without any cross-reactions on the DNA of nonspecific strains. In particular, the sensitivity of this method is down to 0.03125 ng, which is ten times higher than that of real-time PCR. We further evaluate its detection efficiency by testing 15 blood samples from neonatal patients who suffer from pathogen infections, in which some of them have undergone antipathogen treatments. Using the CCP-FRET method, 33.3% (5/15) of samples tested positive for C.albicans and/or K.pneumoniae infections, whereas no pathogen DNAs are recognized with real-time PCR, despite using the same primers. Interestingly, the CCP-FRET method can output unique fluorescent color as well as RGB patterns to different types of pathogen infections, by which the infection type can be conveniently determined. Collectively, the CCP-FRET method is a sensitive and reliable detection platform for rapid identification of fungal and bacterial multiple infections, holding great promise for uses in clinical testing.

Risk of sudden coronary death based on genetic background in Chinese Han population.

Associations between gene variations and sudden cardiac arrest or coronary artery disease have been reported by genome-wide association studies. However, the implication of the genetic status in cases of sudden coronary death (SCD) from the Chinese Han population has remained to be investigated. The present study established a mini-sequencing system to examine putative death-causing single nucleotide polymorphisms (SNPs) using multiplex PCR, single base extension reaction and capillary electrophoresis techniques. A total of 198 samples from the Chinese Han population (age range, 34-71 years; mean age, 53.86 years) were examined using this method. Samples were classified into three groups: Coronary heart disease (CHD, n=70), SCD (n=53) and control (n=75) group. Significant associations were identified for 10, 4 and 6 SNPs in CHD, SCD and sudden death from CHD, respectively, using the χ2 test. The SNPs obtained by binary logistic regression may be used to assess and predict the risk of disease. The predictive accuracy of the SNPs in each prediction model and their area under the receiver operating characteristic curve (AUC) values were determined. The AUC of the four SNPs (rs12429889, rs10829156, rs16942421 and rs12155623) to predict CHD was 0.928, the AUC of the six SNPs (rs2389202, rs2982694, rs10183640, rs597503, rs16942421 and rs12155623) to predict SCD was 0.922 and the AUC of the four SNPs (rs16866933, rs4621553, rs10829156 and rs12155623) to predict sudden death from CHD was 0.912. The multifactor dimensionality reduction values were as follows: 0.8690 (prediction model of CHD), 0.7601 (prediction model of SCD) and 0.7628 (prediction model of sudden death from CHD). Taken together, the results of the present study suggested that these SNPs have considerable potential for application in genetic tests to predict CHD or SCD. However, further studies are required to investigate the putative functions of these SNPs.

A simple and rapid approach for human herpesvirus type 8 subtype characterization using single base extension.

Sequence analysis of the ORFK1 of human herpesvirus type 8 (HHV-8) allows the identification of six major subtypes (A-F), which are related to human migrations and the clinical progression of Kaposi's sarcoma. Sequencing and subsequent phylogenetic analysis of ORFK1 is considered to be the most reliable method for HHV-8 genotyping. However, it exhibits challenges and limitations. Herein, we designed and validated a single base extension (SBE) protocol for characterization of HHV-8 ORFK1 subtypes. A nested polymerase chain reaction (PCR) protocol was carried out to amplify a small 294-bp PCR product encompassing four single nucleotide polymorphisms at positions 360, 406, 465 and 527 of the HHV-8 genome. Finally, a multiplex SBE technique was developed and validated in 20 samples previously genotyped by phylogenetic analysis. The patterns obtained in this reaction could successfully discriminate between ORFK1 subtypes. The typing results obtained completely matched with those of the 'gold standard' method in all analysed samples. This method can reliably identify HHV-8 subtypes A, B and C, which are the most prevalent ones worldwide, and the remaining subtypes (D, E and F). SBE can be useful as an efficient, rapid and low-cost screening method for viral genotyping in a single tube, particularly samples with low-quality DNA, and with easy data interpretation.

Development of a multiplex methylation-sensitive restriction enzyme-based SNP typing system for deconvolution of semen-containing mixtures.

The identification of mixed stains has always been a difficult problem in personal identification in the forensic field. In recent years, tissue-specific methylation sites have proven to be very stable biomarkers for distinguishing tissue origin. However, it is still challenging to perform tissue source identification and individual identification simultaneously. In this study, we developed a method that uses tissue-specific methylation markers combined with single-nucleotide polymorphism (SNP) markers to detect semen from mixed biofluids and to identify individuals simultaneously. Semen-specific CpG markers were chosen from the literature and further validated utilizing methylation-sensitive restriction endonuclease (MSRE) combined with PCR technology. The neighboring SNP markers were searched in the flanking sequence of the target CpG within 400bp, and SNP typing was then carried out through a single-base extension reaction followed by capillary electrophoresis. Eventually, a method of MSRE combined with SNaPshot that could detect 12 compound CpG-SNP markers was developed. Using this system, 10ng of total DNA and DNA mixture with semen content up to 25% could be typed successfully. Moreover, the cumulative discrimination power of the system in the northern Chinese Han population is 0.9998. This study provides a valuable strategy for forensic practice to perform tissue origin and individual identification from mixed stains simultaneously.

A forensic case study for body fluid identification using DNA methylation analysis.

Recently, a method of identifying body fluids using DNA methylation has been developed (Frumkin et al., 2011). An existing multiplex assay using 9 CpG markers could differentiate 5 body fluids: semen, blood, saliva, menstrual blood, and vaginal fluid. To validate this technique, we evaluated the previously described body fluid identification method by means of single base extension (SBE). DNA methylation was applied to 22 samples in 18 forensic cases; seven of these were semen, three were blood, eight were saliva, three were vaginal fluid, and one was menstrual blood. Total of 18 samples were tested, the DNA methylation profiles were coincident from preliminary tests (acid phosphatase (AP), leucomalachite green (LMG, Sigma Aldrich, St Louis, MO, USA) and SALIgAE®) except one sample which displayed an all-negative result. After applying the DNA methylation method to forensic samples, we determined that it could be very useful for differentiating vaginal secretions from menstrual blood, for which there is no conventional preliminary testing method.

Rapid and simple SNP genotyping for Bordetella pertussis epidemic strain MT27 based on a multiplexed single-base extension assay

Multilocus variable-number tandem repeat analysis (MLVA) is widely used for genotyping of Bordetella pertussis, the causative bacteria for pertussis. However, MLVA genotyping is losing its discriminate power because prevalence of the epidemic MT27 strain (MLVA-27) is increasing worldwide. To address this, we developed a single nucleotide polymorphism (SNP) genotyping method for MT27 based on multiplexed single-base extension (SBE) assay. A total of 237 MT27 isolates collected in Japan during 1999–2018 were genotyped and classified into ten SNP genotypes (SG1 to SG10) with a Simpson’s diversity index (DI) of 0.79 (95% CI 0.76–0.82). Temporal trends showed a marked increase in the genotypic diversity in the 2010s: Simpson’s DI was zero in 1999–2004, 0.16 in 2005–2009, 0.83 in 2010–2014, and 0.76 in 2015–2018. This indicates that the SNP genotyping is applicable to the recently circulating MT27 strain. Additionally, almost all outbreak-associated MT27 isolates were classified into the same SNP genotypes for each outbreak. Multiplexed SBE assay allows for rapid and simple genotyping, indicating that the SNP genotyping can potentially be a useful tool for subtyping the B. pertussis MT27 strain in routine surveillance and outbreak investigations.

Characterization of single nucleotide polymorphism in IGF1 and IGF1R genes associated with growth traits in striped catfish (Pangasianodon hypophthalmus Sauvage, 1878)

Development of molecular markers for growth trait in striped catfish (Pangasianodon hypophthalmus) is of importance to the aquaculture industry in the Mekong river delta. This study aims to analyze the genetic variation and the effects of the Insulin-like growth factor 1 (IGF1) and Insulin-like growth factor 1 receptor (IGF1R) genes on growth traits in striped catfish. We sequenced fragments of IGF1 and IFG1R genes from 10 individuals of fast-growing fish and 10 individuals of slow-growing fish to discover single nucleotide polymorphisms (SNPs). Among the discovered SNPs, one SNP (13,680 A > T in intron 2) of IGF1 gene, three SNPs (13,357 T > C, 15392 T > A in intron 1 and 83,894 A > G in intron 15) in IGF1R gene were filtered and further validated by individual genotyping in 70 fast-growing fish and 70 slow-growing fish using the multiplex SNaPshot method (single base extension method). Our analyses showed that two SNPs (IGF1:13680 A > T and IGF1R:13357 T > C) were significantly associated with the growth trait (p < 0.01 and p < 0.05, respectively) and therefore considered as candidate SNP markers for growth trait in P. hypophthalmus. In addition, a haplotype analysis was carried out to confirm the association between three SNPs in IGF1R gene with growth traits. Moreover, the combination of the two candidate SNP markers was further analyzed to examine their cumulative effect on the growth traits. The result showed that the combination of genotype AA of IGF1:13680 A > T and genotype TT of IGF1R:13357 T > C led to the positive cumulative effect on the growth (p < 0.01). The variations identified in this study have a potential to be marker candidates for assisted selection in genetic breeding of the striped catfish.