Ng Of DNA Research Articles

Alternative direct-to-amplification sperm cell lysis techniques for sexual assault sample processing.

The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although traditional DNA extraction and purification utilizing detergents, proteinase K, and DTT have been the primary technique for lysing sperm cell fractions from these samples, it is labor‐intensive and inefficient regarding time and sperm DNA recovery – hindering the ability of forensic analysts to keep pace with evidence submissions. Thus, this study examined seven alternative sperm cell lysis techniques to develop a method that could efficiently lyse sperm and consistently generate high‐quality profiles while also reducing time, labor, and cost requirements. Microscopic examination of lysates indicated only Casework Direct and alkaline techniques could lyse all spermatozoa within samples, while quantification results demonstrated all methods performed comparably to the control method of forensicGEM™ Sperm (p > 0.06). Amplification with 0.25 ng DNA revealed that unpurified lysates from Casework Direct, alkaline, and NP‐40 techniques produced DNA profiles with acceptable mean STR peak heights and interlocus balance, both of which were similar to or better than the control. Overall, this study demonstrated the ability of Casework Direct, alkaline, and NP‐40 methods to efficiently lyse spermatozoa and provide high‐quality STR profiles despite the absence of a purification step. Ultimately, based on the data reported herein, alkaline lysis is the recommended alternative sperm lysis approach given its ability to generate high‐quality profiles, save time, and decrease the cost per reaction when compared to traditional sperm cell lysis methods.

Whole Genome DNA Methylation Profiling of D2 Medium Spiny Neurons in Mouse Nucleus Accumbens Using Two Independent Library Preparation Methods.

DNA methylation plays essential roles in various cellular processes. Next-generation sequencing has enabled us to study the functional implication of DNA methylation across the whole genome. However, this approach usually requires a substantial amount of genomic DNA, which limits its application to defined cell types within a discrete brain region. Here, we applied two separate protocols, Accel-NGS Methyl-Seq (AM-seq) and Enzymatic Methyl-seq (EM-seq), to profile the methylome of D2 dopamine receptor-expressing medium spiny neurons (D2-MSNs) in mouse nucleus accumbens (NAc). Using 40 ng DNA extracted from FACS-isolated D2-MSNs, we found that both methods yielded comparably high-quality methylome data. Additionally, we identified numerous unmethylated regions (UMRs) as cell type-specific regulatory regions. By comparing the NAc D2-MSN methylome with the published methylomes of mouse prefrontal cortex excitatory neurons and neural progenitor cells (NPCs), we identified numerous differentially methylated CpG and non-CpG regions. Our study not only presents a comparison of these two low-input DNA whole genome methylation profiling protocols, but also provides a resource of DNA methylome of mouse accumbal D2-MSNs, a neuron type that has critical roles in addiction and other neuropsychiatric disorders.

Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing

BackgroundNovel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits.ResultsWe compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits’ fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection.ConclusionsThe enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Application of family-specific primers in multiplex real-time PCR for meat categories screening

Meat adulteration has been a quality issue in entire meat supply chain, which can harm consumers’ economic and health interests seriously. In order to prevent adulteration of mutton and reduce the cost of testing, three family-specific primers were designed and then a triplex real-time PCR method with melting curve analysis was developed, which could identify three meat categories simultaneously. The primers were designed from mitochondrial gene including 16S rRNA and 12S rRNA. The primer CMRC-Ovis/Capra3 can only recognize Ovis category including sheep and goat without recognizing other species. Similarly, primer CMRC-Canidae2 can only identify Canidae category including dog, fox and racoon-dog; and CMRC-Poultry3 can only identify poultry category including chicken, duck and goose respectively. The amplicons’ melting temperature of three categories were 72.45∼75.91 °C, 77.25∼78.76 °C and 84.58∼87.40 °C, respectively. The specificity, universality, detection limit and practicability were validated and the results showed that all family-specific primers have good specificity and universality. This method has low detection limit (0.001∼0.1 ng DNA), strong reproducibility, and good practicability, which indicated a good application prospect in the screening of adulterated species in mutton products.

Internal validation for analytical and stochastic thresholds in the GlobalFiler system

Interpretation of crime stain profiles is one of the challenging tasks of forensic scientists. Before using the probabilistic genotyping system, forensic scientists should interpret crime stain profiles using analytical and stochastic thresholds. According to the guidelines presented by the Scientific Working Group on DNA Analysis Methods, these thresholds shall be based on and supported by applicable internal validation studies. In this study, we performed the internal validation study of our DNA typing system to determine these thresholds. A total of 350 DNA samples and 11 negative controls were amplified using GlobalFilerTM PCR Amplification Kit with 30 cycles and PCR products were then analyzed on a SeqStudio Genetic Analyzer. As a result, the analytical threshold was set to 110 RFU, which has high sensitivity to obtain almost full profiles of 0.063 ng DNA, and has high specificity not to detect most of the pull-up and drop-in peaks. The stochastic threshold was set to 890 RFU allowing a 1% probability of allelic drop-out based on logistic regression. These thresholds are useful for interpreting crime stain profiles in our GlobalFiler system.

Single-Tube qPCR Detection and Quantitation of Hotspot Mutations Down to 0.01% Variant Allele Fraction.

Clinically and biologically, rare DNA sequence variants are significant and informative. However, existing common detection technologies are either complex and time-consuming in workflow, or restricted in the limit of detection (LoD), or do not allow for multiplexing. Blocker displacement amplification (BDA) method can stably and effectively detect and enrich multiple rare variants with LoD around 0.1% variant allele fraction (VAF). Nonetheless, the detailed mutation information has to be identified by additional sequencing technologies. Here, we present allele-specific BDA (As-BDA), a method combining BDA with allele-specific TaqMan (As-TaqMan) probes for effective variant enrichment and simultaneous single nucleotide variant or small insertions and deletions (INDELs) profiling. We demonstrated that As-BDA could detect mutations down to 0.01% VAF. Further, As-BDA could detect up to four mutations with low to 0.1% VAF per reaction using only 15 ng DNA input. The median error of As-BDA in VAF determination is approximately 9.1%. Comparison experiments using As-BDA and droplet digital PCR on peripheral blood mononuclear cell clinical samples showed 100% concordance for samples with mutations at ≥ 0.1% VAF. Hence, we have shown that As-BDA can achieve simultaneous enrichment and identification of multiple targeted mutations within the same reaction with high clinical sensitivity and specificity, thus helpful for clinical diagnosis.

Fetal Nucleated Red Blood Cells Preferable Than Cell-Free Fetal DNA for Early Determination of Gender Among Invasive and Non-Invasive Source Using Novel Four Genes Multiplex PCR.

BackgroundDeoxyribonucleic acid from invasive, non-invasive and 9th week embryo can be a resource for the determination of fetal sex using highly sensitive and specific multiplex PCR.MethodsA total of 402 DNA samples were used to test the newly developed novel multiplex PCR including male specific (3 genes: SRY, DAZ2 and TSPY1) Y-biomarkers and internal control, ACTB. The study isolated cffDNA (Cell-free fetal DNA; n = 73) from mother’s plasma, serum and urine, fetal DNA from 9th week embryo and cord blood, and fetal DNA from CD71+ve nucleated red blood cells (fNRBC; n = 73). Paternal and maternal DNA from buccal cells (n = 20) and blood (n = 232) used for male and female confirmation.ResultsThe study observed that SRY alone cannot be a suitable Y-biomarker. Confirmation from any two Y-biomarkers is mandatory for male fetus identification. Direct sequencing of the gel eluted multiplex and single amplicons confirmed the specific sequences. Presence of two out of 3 Y-biomarkers OR single Y-biomarker with >1,000,000 intensity is considered positive for male. The multiplex PCR is suitable for determining sex from all source of fetal DNA including highly degraded cffDNA and can detect the sex using 0.5ng DNA. Individual marker-based real-time qPCR followed by combined melt curve analysis showed distinguished melt curve peaks for the markers.ConclusionThe multiplex PCR achieved 100% accuracy on fetal DNA from fNRBC for early determinations (<13 weeks) of gender. The developed novel and simple multiplex PCR and individual qPCR can be adopted in all types of laboratories for determining human fetal gender using fetal DNA from fNRBC. Early identification of gender can support to prepare for possible X-linked analysis, reduce anxiety in mother, strengthen a bond between mother and fetus, and effective decision making. Non-invasive source of fetal DNA from fNRBC preferred for identifying gender to reduce the risk of invasive procedures in early (8–13 weeks) pregnancy.

Detection and Real-time PCR Assay for the Quantification of Carbapenemase Gene blablaNDM-1 in Hospital Effluent

This study aims to isolate gram-negative bacteria (GNB) harboring the gene NDM-1 from the tertiary care hospital effluents. Also, aims to evaluate the relative copy number of blaNDM-1 carried by the positive isolates. The study isolated 215 GNB from 40 effluent samples. The antibiotic susceptibility tests for carbapenems were performed using disc diffusion assay. The isolates resistant to either meropenem or imipenem were checked for the existence of MBL by phenotypic methods. The isolates carrying NDM-1 gene were genotypically confirmed by Polymerase chain reaction (PCR). The gene copy number of blaNDM- were determined by quantative real-time PCR. A total of 22 isolates showed phenotypic resistance to carbapenems and were characterized by biochemical methods. Among them, 12 harbored NDM-1 gene by PCR; these bacteria were subjected to qPCR for determining the absolute copy numbers of the NDM-1 gene on it. The gene abundance in the strains was in the range of 3.28× 105 to 6.05× 106 copies/ ng of DNA. Hospital effluents are important pool of antibiotic-resistant bacteria harboring the blaNDM-1 and infections caused by these bacteria are difficult to treat. Hence, the present study stresses the need for stringent antibiotic use and efficient wastewater treatment policies in these hospital settings, which is paramount in achieving sustainable health.

Evaluation of a Rapid Automated Next Generation Sequencing Assay for Precision Medicine in Acute Myeloid Leukemia

Background:In the last decade there have been significant advances in diagnosing and classifying adult acute myeloid leukemia (AML) based on genomic profiling, enabling risk-stratification and targeted therapies. In 2017 the US FDA approved the first gene mutation targeted therapies for AML with multiple additional targeted therapies since approved or in development. Given the typical acuity of AML at initial presentation however and the current turnaround time for next-generation sequencing (NGS) assays, most patients will start definitive initial therapy before all potentially targetable mutations are known. There is, therefore, a significant need for a fast molecular genotyping test to determine eligibility for personalized therapy in AML.The NCI Myeloid Assay (NMA) is a comprehensive targeted NGS assay on the Ion Torrent Genexus System, a fully automated platform that provides a rapid turnaround time from specimen receipt to clinical reporting. NMA utilizes Thermo Fisher Scientific's Oncomine Myeloid Assay GX and appears ideally suited for use in upcoming AML targeted therapy trials but has yet to be extensively tested in a cohort of AML patient diagnostic samples and compared to a standard targeted “myeloid panel” NGS assay platform (s-NGS).Methods:DNA samples (n=173) extracted from pretreatment bone marrow and/or peripheral blood of adult patients (n=112) diagnosed with de novo AML or high-risk myelodysplastic syndrome (MDS), were blindly tested in parallel using the NMA and s-NGS assays. For the NMA assay, 27.75ng of DNA was put into the Genexus System. All runs, controls, and samples were first analyzed for sequencing quality using established quality control (QC) metrics to assess pass/fail status. For all samples that passed QC metrics, variant results generated by the Ion Torrent Genexus pipeline were manually reviewed prior to being called true positive variants. For the s-NGS, using the ArcherDx Myeloid VariantPlex assay, a DNA input of 50ng was used for library preparation on a dual pre- and post-PCR separated automated liquid-handling workflow. Resulting libraries were sequencing on the Novaseq 6000 (Illumina) and the data analyzed using the Archer Analysis software and filtered as previously described (PMID: 34258102). Results from the two assays were compared for mutations with a variant allele fraction (VAF) >5% occurring in genes of interest in small molecule targeted clinical trials including: FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, KRAS, and TP53. For FLT3-ITD comparison, the presence or absence of a call by the assay was used.Results:Utilizing a 5% VAF reporting threshold, a total of 171 and 174 variants were detected by NMA and s-NGS assays, respectively. A high rate of concordance was observed between the assays, with NMA detecting 96% of s-NGS variants and s-NGS detecting 95% of NMA variants. The VAF of detected single nucleotide variants was highly correlated (r=0.9848, P<0.0001, Figure 1A). NPM1 mutation VAF values trended lower by s-NGS compared to NMA.We investigated the discordant calls (n=15 total in 11 patients). One patient was correctly identified as having an NRAS p.Gly12 mutation by both approaches, but the resulting mutation was incorrectly annotated by the s-NGS pipeline. Samples from two patients (including one with both blood and marrow tested) were correctly identified as being FLT3 tyrosine kinase domain mutated by both sequencing approaches, although only the major of two missense variants identified by s-NGS was reported by the NMA pipeline. None of these patients, however, would be misclassified. The remaining 11 discordant calls were false negatives (including 6 variants detected by s-NGS but not by NMA). All of these “edge case” variants were detectable by lowering the VAF reporting threshold below 5% (Figure 1B).Conclusions:NMA is an automated sample-to-results workflow that can identify myeloid disorder-associated genomic variants in less than 48 hours from library preparation to clinical reporting. We show that NMA is highly concordant with a standard DNA NGS assay for detecting mutations within recurrently mutated AML genes. Accurate rapid genotyping is required for assignment to initial treatment with targeted therapy, and this technology may be a valuable tool for upcoming clinical trials for patients with myeloid malignancies. [Display omitted] DisclosuresZhang: Thermo Fisher Scientific: Current Employment. Sedova: Thermo Fisher Scientific: Current Employment. Huang: Thermo Fisher Scientific: Current Employment. Mittal: Thermo Fisher Scientific: Current Employment. Hatch: Thermo Fisher Scientific: Current Employment. Ni: Thermo Fisher Scientific: Current Employment. Kaznadzey: Thermo Fisher Scientific: Current Employment. Sadis: Thermo Fisher Scientific: Current Employment. Smith: Thermo Fisher Scientific: Current Employment. Williams: Illumina: Other: CRADA. Hourigan: Sellas: Research Funding.

Pediatric Patients with Immune Thrombocytopenic Purpura Have a Dysbiotic Gut Microbiome at Time of Diagnosis

Background: Pediatric immune thrombocytopenia purpura (ITP) is the most common cause of autoimmune cytopenias in children and results in autoimmune destruction of platelets, leading to significantly decreased circulating platelets, increased bleeding risk and fatigue. The underlying mechanism of action is thought to be auto-antibody driven, though auto-reactive T cells have also been implicated in some cases. Given the proximity of many cases of ITP to either recent viral infection or vaccine administration, a leading hypothesis is that the environmental exposure mimics a platelet produced epitope causing platelet clearance. Despite the correlation of viral infection and/or inflammatory cascades in the propagation of ITP, the gut microbiome in newly diagnosed pediatric patients has not previously been interrogated. The gut microbiome has previously been shown to modulate the host inflammatory milieu and dysbiosis has been associated with other auto-immune disorders. Thus, we assessed the gut microbiome in newly diagnosed pediatric ITP patients to determine if a similar dysbiosis is present.Methods: Stool samples were collected from 32 pediatric patients (0-18 years) at the Children's Hospital of Philadelphia. 17 patients were newly diagnosed with ITP, 7 patients had ITP for greater than 3 months at time of stool collection and 8 patients had newly diagnosed severe acquired aplastic anemia. Stool samples were kept on ice until processing at our PENN/CHOP Microbiome Core (not longer than 24 hours/sample). Age matched healthy control samples were provided by the Microbiome Core. Shotgun libraries were generated from 0.5 ng DNA using the Nextera XT Library Prep kit and libraries were sequenced on an Illumina HiSeq 2500 in High Output mode to produce paired-end 125 bp sequence reads. Shotgun metagenomic data were analyzed using Sunbeam, a user-extendable bioinformatics pipeline that we developed for this purpose.Diversity within samples were assessed by the number of OTUs at a rarefaction level of 1,000 sequences and the Shannon index. Sample similarity were assessed by Bray-Curtis and Jaccard distances, which were then visualized using principle coordinates analysis.Results: Subjects included 17 patients with newly diagnosed ITP (i.e. stool sample collected < 3 months from diagnosis), 7 patients with ITP > 3 months and 8 patients with newly diagnosed severe acquired aplastic anemia. Patient samples were assessed for gut microbial diversity and richness. As seen in figure 1a, gut microbial diversity in newly diagnosed pediatric patients with ITP was significantly decreased (p = 0.024) while gut microbial richness trended to a concomitant decrease as well (p = 0.093). Interestingly, healthy controls had similar gut microbiomes to one another than newly diagnosed ITP patients had to one another (Fig 1b). These alterations in the gut microbiome were not seen in pediatric patients with newly diagnosed aplastic anemia or with ITP diagnosed > 3 months prior to collection (data not shown). These findings indicate that while there is a dysbiotic gut microbiome in patients with newly diagnosed ITP, there is no dysbiosis present in other pediatric autoimmune cytopenias. Furthermore, the dysbiosis corrects by 3 months post-ITP diagnosis. When we assess specific bacterial families and subgroups we do not find a predominant species that is increased or decreased but rather there are global changes amongst many bacterial species. We did find a trend of lower Alistipes species and increased Eschrechia coli in newly diagnosed ITP patients which is similar to other autoimmune disorders with shifted gut microbiota. Functional pathways analyses showed similar global alterations in vital pathways such as decreased aminoacyl tRNA biosynthesis and homologous recombination.Conclusion: Pediatric patients with newly diagnosed ITP but not other autoimmune cytopenias have gut microbial dysbiosis with perturbations in many bacterial species that may be a cause of, or a result of the underlying mechanism of pathogenesis. Further studies will help determine the role of dysbiosis in the pathobiology of ITP and determine whether intervention alters the duration of disease. [Display omitted] DisclosuresLambert: Bayer: Consultancy; Astra Zeneca: Research Funding; Shionogi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ClinGen, ISTH, ASH, GW University: Honoraria; PDSA: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dova: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Clinically Validated Targeted Capture Panel to Identify Translocations, Copy Number Abnormalities, and Mutations in Multiple Myeloma

A Clinically Validated Targeted Capture Panel to Identify Translocations, Copy Number Abnormalities, and Mutations in Multiple Myeloma

A novel co-amplification system for simultaneous amplification of 23 Y-STR and identification of spermatozoa.

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80ng/0.1ng) or a mix of the menstrual blood/semen DNA (5ng/0.1ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1ng semen DNA, even in the presence of 5ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases.

Estimation of economic loss by carcass weight reduction of Japanese dairy cows due to infection with bovine leukemia virus

Bovine leukemia virus (BLV) infection is endemic in Japanese dairy farms. To promote the participation of farmers in BLV infection control in Japan, it is important to provide estimates of the economic losses caused by this infection. We hypothesized that decreased immune function due to BLV infection would increase visceral abnormalities, in turn reducing carcass weight. We employed mediation analysis to estimate the annual economic loss due to carcass weight reduction caused by BLV infection.Culled Holstein cows from 12 commercial dairy farms in the Nemuro and Kushiro regions of Hokkaido, Japan, were traced. Information on age and the last delivery day were collected. A non-infected culled cow was defined as a cow from which BLV provirus was not detected. A high-proviral-load (H-PVL) cow was defined as a cow whose PVL titer was above 2465 copies/50 ng DNA or 56,765 copies/105 cells. A BLV-infected cow with PVL titer lower than the thresholds was categorized as low-proviral load (L-PVL). Post-mortem examination results for culled cows were collected from a meat inspection center.The hypothesis was tested by three models, using data from 222 culled dairy cows. Model 1, a generalized linear mixed-effects model, selected carcass weight as an outcome variable, BLV status and the potential confounders (lactation stage and age) as explanatory variables, and herd as a random effect. Model 2 additionally included the number of abnormal findings in the post-mortem examination (AFPE) as an explanatory variable. Model 3 applied a Bayesian generalized linear mixed model, which employed a mediator separately modeled for AFPE, to estimate the amount of direct, indirect, and total carcass weight loss with adjustment for known confounding factors.Compared to the mean carcass weight for the non-infected culled cows, the carcass weight for H-PVL culled cows was significantly decreased by 30.4 kg on average. For each increase of one in the number of AFPE, the mean carcass weight was decreased by 8.6 kg. Only the indirect effect of BLV H-PVL status on carcass weight loss through AFPE was significant, accounting for 21.6 % of the total effect on carcass weight reduction. In 2017, 73,650 culled dairy cows were slaughtered in Hokkaido, and the economic loss due to carcass weight loss caused by BLV infection that year was estimated to be US $1,391,649. In summary, unlike L-PVL cows, H-PVL status was associated with carcass weight reduction, which was partially mediated by an increase in the number of visceral abnormalities.

Evaluation of a custom QIAseq targeted DNA panel with 164 ancestry informative markers sequenced with the Illumina MiSeq

Introduction of new methods requires meticulous evaluation before they can be applied to forensic genetic case work. Here, a custom QIAseq Targeted DNA panel with 164 ancestry informative markers was assessed using the MiSeq sequencing platform. Concordance, sensitivity, and the capability for analysis of mixtures were tested. The assay gave reproducible and nearly concordant results with an input of 10 and 2 ng DNA. Lower DNA input led to an increase in both locus and allele drop-outs, and a higher variation in heterozygote balance. Locus or allele drop-outs in the samples with less than 2 ng DNA input were not necessarily associated with the overall performance of a locus. Thus, the QIAseq assay will be difficult to implement in a forensic genetic setting where the sample material is often scarce and of poor quality. With equal or near equal mixture ratios, the mixture DNA profiles were easily identified by an increased number of imbalanced heterozygotes. For more skewed mixture ratios, the mixture DNA profiles were identified by an increased noise level. Lastly, individuals from Great Britain and the Middle East were investigated. The Middle Eastern individuals showed a greater affinity with South European populations compared to North European populations.

Characterization of innately decellularised micropattern pseudostem of Musa balbisiana - A non-surface functionalized 3D economic biomaterial scaffold

Banana (Musa balbisiana) pseudostem 3D scaffolds have been developed here for primary eukaryotic cell and cell line culture as an economical, sustainable, eco-friendly alternative for surface-functionalized polymeric and plant tissue-based structures. Musa pseudostem 3D micro pattern scaffold (MPM-3Ds) developed by freeze-drying followed by ethylene oxide sterilization yielded 5.6ng of DNA per mg of tissue, confirming its extended decellularised state. Thermogravimetric analysis, contact angle measurement, uniaxial testing, and FTIR determined thermal stability, wettability, tensile strength, and surface functional groups respectively. Micro and macronutrients, sugars, and amino acids that naturally enrich MPM-3Ds were estimated using EDAX, HPLC, and biochemical analysis. The most important finding was, non-surface functionalized MPM-3Ds supported attachment, growth, and differentiation of human mesenchyme stem cells, human primary hepatocytes like cells, primary mouse brain cortical neurons, mouse fibroblast cells, and human pancreatic cancer cells. MPM-3Ds showed in vivo biodegradation and biocompatibility in a preliminary analysis in Sprague Dawley rats. These findings illuminate nature's power to nurture cells in the micropattern cradles of MPM- 3Ds that can support innovative research in stem cell differentiation, drug and cosmetic testing, and biosensor development leading to advanced biomedical research.

Characterization of innately decellularised micropattern pseudostem of Musa balbisiana - A non-surface functionalized 3D economic biomaterial scaffold

Banana (Musa balbisiana) pseudostem 3D scaffolds have been developed here for primary eukaryotic cell and cell line culture as an economical, sustainable, eco-friendly alternative for surface-functionalized polymeric and plant tissue-based structures. Musa pseudostem 3D micro pattern scaffold (MPM-3Ds) developed by freeze-drying followed by ethylene oxide sterilization yielded 5.6ng of DNA per mg of tissue, confirming its extended decellularised state. Thermogravimetric analysis, contact angle measurement, uniaxial testing, and FTIR determined thermal stability, wettability, tensile strength, and surface functional groups respectively. Micro and macronutrients, sugars, and amino acids that naturally enrich MPM-3Ds were estimated using EDAX, HPLC, and biochemical analysis. The most important finding was, non-surface functionalized MPM-3Ds supported attachment, growth, and differentiation of human mesenchyme stem cells, human primary hepatocytes like cells, primary mouse brain cortical neurons, mouse fibroblast cells, and human pancreatic cancer cells. MPM-3Ds showed in vivo biodegradation and biocompatibility in a preliminary analysis in Sprague Dawley rats. These findings illuminate nature's power to nurture cells in the micropattern cradles of MPM- 3Ds that can support innovative research in stem cell differentiation, drug and cosmetic testing, and biosensor development leading to advanced biomedical research.

Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.

Diagnóstico de ectima contagioso em pequenos ruminantes através da Reação em Cadeia da Polimerase em Tempo Real

The virus of contagious ecthyma (CEV), also known as orf virus (ORFV) is the etiological agent of contagious ecthyma (CE) in sheep and goat and belongs to the Parapoxvirus genus, family Poxviridae. In some cases, CE can be confused with vesicular diseases so there is need for differentiation especially because, according to the standards of the National Program for the Eradication of FMD (PNEFA), goats and sheep are not vaccinated against Foot and Mouth Disease (FMD), acting as sentinel animals. Although initial studies have demonstrated the usefulness of the polymerase chain reaction (PCR) as a diagnostic test, there are no studies involving its use on Brazilian field samples, which may be genetically distinct from previously studied samples, as described in a study of restriction sites analysis of Brazilian CE samples. This work was conducted with the goal of standardizing a PCR (qPCR) test using SYBR Green I dye for molecular diagnosis of EC in DNA extracted from lesions of affected animal or cell culture inoculated in field samples. The products were detected with qPCR dissociation curve analysis which showed a peak at 88 ºC indicating that positive samples have only one specific amplification product. All DNA samples tested (29 animals crusts and their cell cultures) were positive in the qPCR. The qPCR was able to detect the DNA of at least 10,000 times dilution corresponding to 0.056 ng of DNA. It is believed that with the additional qPCR validations reported in this study, it can be used for differential diagnosis in the health surveillance of PNEFA.

Standardization of DNA amount for bisulfite conversion for analyzing the methylation status of LINE-1 in lung cancer.

Highly methylated Long Interspersed Nucleotide Elements 1 (LINE-1) constitute approximately 20% of the human genome, thus serving as a surrogate marker of global genomic DNA methylation. To date, there is still lacking a consensus about the precise location in LINE-1 promoter and its methylation threshold value, making challenging the use of LINE-1 methylation as a diagnostic, prognostic markers in cancer. This study reports on a technical standardization of bisulfite-based DNA methylation analysis, which ensures the complete bisulfite conversion of repeated LINE-1 sequences, thus allowing accurate LINE-1 methylation value. In addition, the study also indicated the precise location in LINE-1 promoter of which significant variance in methylation level makes LINE-1 methylation as a potential diagnostic biomarker for lung cancer. A serial concentration of 5-50-500 ng of DNA from 275 formalin-fixed paraffin-embedded lung tissues were converted by bisulfite; methylation level of two local regions (at nucleotide position 300–368 as LINE-1.1 and 368–460 as LINE-1.2) in LINE-1 promoter was measured by real time PCR. The use of 5 ng of genomic DNA but no more allowed to detect LINE-1 hypomethylation in lung cancer tissue (14.34% versus 16.69% in non-cancerous lung diseases for LINE-1.1, p < 0.0001, and 30.28% versus 32.35% for LINE-1.2, p < 0.05). Our study thus highlighted the optimal and primordial concentration less than 5 ng of genomic DNA guarantees the complete LINE-1 bisulfite conversion, and significant variance in methylation level of the LINE-1 sequence position from 300 to 368 allowed to discriminate lung cancer from non-cancer samples.

Response of soil bacterial community to agricultural reclamation in the Tengger desert, northwestern China

Given the conditions of scarce soil water availability, high UV exposure and huge temperature fluctuation, plant growth in desert is extremely limited. In the last three decades, reclaiming desertified land for agricultural use via the application of solar greenhouse has been developing in the arid regions, northwestern China. However, the soil microbiome shift driven by the transformation of land use remains largely unexplored. To this end, we examined the diversity and composition of the bacterial community in the vineyard soil at the edge of Tengger desert, using the high-throughput quantitative 16S rRNA gene sequencing. Two types of agricultural managed soils, including those near the plant (PLT) and the others on the fertilization furrow (FTL), were collected. Soils from the open desert land without agricultural practices were used as the control (CON). The results showed that agricultural reclamation significantly (p < 0.05) reduced the desert soil bacterial diversity while promoted the total bacterial abundance from 106 copies up to 108 per ng of DNA, which increased up to 90 folds. Among the dominant bacteria Firmicutes, Proteobacteria, Actinobacteria, and Acidobacteria, Bacilli and Actinobacteria were found to be the keystone taxa responding to the agricultural practices especially to the nutrient inputs from fertilization. The functional prediction of the microbiome suggested that amino acid and carbohydrate transport and metabolism were the most important metabolic pathways differed between the three types of soils. Overall, our findings provide deep insights into the understanding of desert soil microbial responses to agricultural reclamation on the processes of human urbanization and reversion of desertification.