Detection Of Single Nucleotide Polymorphisms Research Articles

Detection of vkorc1 polymorphism: comparison of polymerase chain reaction/restriction fragment length polymorphism (pcr + rflp) with allele–specific polymerase chain reaction

Purpose: The VKORC1 polymorphism is an important genetic factor affecting warfarin dose requirement. Patients require different warfarin doses in order to achieve the target therapeutic anticoagulation. The aim of our study was to determine the frequency of single nucleotide polymorphisms (SNP) in the VKORC1 gene in the general population, using a simple, rapid, and economical method.
 Methods: For genotyping, the restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)–amplified DNA was used and compared to allele– specific polymerase chain reaction. We genotyped 441 DNA samples obtained from the healthy general population in North–Eastern Slovenia. Genotypes for the tested group were evaluated to determine whether the population followed the Hardy–Weinberg equilibrium. The genotypes and allele frequencies were calculated.
 Results: The results obtained using the allele–specific polymerase chain reaction were consistent with those obtained using the PCR + RFLP method. The G allele frequency (0.62) was higher than the A allele frequency (0.38) in the general population from North–Eastern Slovenia.
 Conclusions: The PCR+RFLP method involved additional manipulation of the PCR products at the expense of analysis time, consumption of reagents and equipment. The allele–specific polymerase chain reaction was a simple and rapid method for the detection of SNP in the VKORC1 gene, and is available in any laboratory with the minimum of equipment and reagents required.

DNA Genotyping Based on Isothermal Amplification and Colorimetric Detection by Consumer Electronics Devices.

The point-of-care testing of DNA biomarkers requires compact biosensing systems and consumer electronic technologies provide fascinating opportunities. Their portability, mass-produced components, and high-performance readout capabilities are the main advantages for the development of novel bioanalytical methods.This chapter describes the detection of single nucleotide polymorphisms (SNP) through methods based on user-friendly optical devices (e.g., USB digital microscope, flatbed scanner, smartphone, and DVD drive). Loop mediated isothermal amplification (LAMP) enables the required discrimination of each specific variant prior to the optical reading. In the first method, products are directly hybridized to the allele-specific probes attached to plastic chips in an array format. The second method, allele-specific primers are used, enabling the direct end-point detection based a colorimetric dyer and a microfluidic chamber chip. In both approaches, devices are employed for chip scanning.A representative application to the genotyping of a clinically relevant SNP from human samples is provided, showing the excellent features achieved. Consumer electronic devices are able to register sensitive precise measurements in terms of signal-to-noise ratios, image resolution, and scan-to-scan reproducibility. The integrated DNA-based method lead a low detection limit (100genomic DNA copies), reproducible (variation <15%), high specificity (genotypes validated by reference method), and cheap assays (<10€/test). The underlying challenge is the reliable implementation into minimal-specialized clinical laboratories, incorporating additional advantages, such as user-friendly interface, low cost, and connectivity for telemedicine needs.

Universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR) for SNP genotyping

BackgroundThe detection and identification of single nucleotide polymorphism (SNP) is essential for determining patient disease susceptibility and the delivery of medicines targeted to the individual. At present, SNP genotyping technology includes Sanger sequencing, TaqMan-probe quantitative polymerase chain reaction (qPCR), amplification-refractory mutation system (ARMS)-PCR, and Kompetitive Allele-Specific PCR (KASP). However, these technologies have some disadvantages: the high cost of development and detection, long and time consuming protocols, and high false positive rates. Focusing on these limitations, we proposed a new SNP detection method named universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR). In this method, only two types of fluorescence-labeled probes were used for SNP genotyping, thus greatly reducing the cost of development and detection for SNP genotyping.ResultsIn the amplification process of UPIP-qPCR, unlabeled intermediate primers with template-specific recognition functions could trigger probe hydrolysis and specific signal release. UPIP-qPCR can be used successfully and widely for SNP genotyping. The sensitivity of UPIP-qPCR in SNP genotyping was 0.01 ng, the call rate was more than 99.1%, and the accuracy was more than 99.9%. High-throughput DNA microarrays based on intermediate primers can be used for SNP genotyping.ConclusionThis novel approach is both cost effective and highly accurate; it is a reliable SNP genotyping method that would serve the needs of the clinician in the provision of targeted medicine.

Solid-phase recombinase polymerase amplification using ferrocene-labelled dNTPs for electrochemical detection of single nucleotide polymorphisms

Hypertrophic cardiomyopathies (HCM) are the principal cause of sudden cardiac death in young athletes and it is estimated that 1 in 500 people have HCM. The aim of this work was to develop an electrochemical platform for the detection of HCM-associated SNP in the Myosin Heavy Chain 7 (MYH7) gene, in fingerprick blood samples. The platform exploits isothermal solid-phase primer elongation using recombinase polymerase amplification with either individual or a combination of four ferrocene-labelled nucleoside triphosphates. Four thiolated reverse primers containing a variable base at their 3’ end were immobilised on individual gold electrodes of an array. Following hybridisation with target DNA, solid phase recombinase polymerase amplification was carried out and primer elongation incorporating the ferrocene labelled oligonucleotides was only detected at one of the electrodes, thus facilitating identification of the SNP under interrogation. The assay was applied to the direct detection of the SNP in fingerprick blood samples from eight different individuals, with the results obtained corroborating with next generation sequencing. The ability to be able to robustly identify the SNP using a 10 μL fingerprick sample, demonstrates that SNP discrimination is achieved using low femtomolar (ca. 8 × 105 copies DNA) levels of DNA.

Low-Cost Platform for Multiplexed Electrochemical Melting Curve Analysis.

Detection and identification of single nucleotide polymorphisms (SNPs) have garnered increasing interest in the past decade, finding potential application in detection of antibiotic resistance, advanced forensic science, as well as clinical diagnostics and prognostics, moving toward the realization of personalized medicine. Many different techniques have been developed for genotyping SNPs, and ideally these techniques should be rapid, easy-to-use, cost-effective, flexible, scalable, easily automated, and requiring minimal end-user intervention. While high-resolution melting curve analysis has been widely used for the detection of SNPs, fluorescence detection does not meet many of the desired requirements, and electrochemical detection is an attractive alternative due to its high sensitivity, simplicity, cost-effectiveness, and compatibility with microfabrication. Herein, we describe the multiplexed electrochemical melting curve analysis of duplex surfaces tethered to electrodes of an array. In this approach, thiolated probes designed to hybridize to a DNA sequence containing the SNP to be interrogated are immobilized on gold electrodes. Asymmetric PCR using a ferrocene-labeled forward primer is used to generate this single-stranded redox-labeled PCR amplicon. Following hybridization with the probe immobilized on the electrode surface, the electrode array is exposed to a controlled ramping of temperature, with concomitant constant washing of the electrode array surface while simultaneously carrying out voltammetric measurements. The optimum position of the site complementary to the SNP site in the immobilized probe to achieve maximum differentiation in melting temperature between wild-type and single base mismatch, thus facilitating allelic discrimination, was determined and applied to the detection of a cardiomyopathy associated SNP.

Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides.

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5′-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2′-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

Analysis of parental contribution for aneuploidy detection (APCAD): a novel method to detect aneuploidy and mosaicism in preimplantation embryos

Research questionCan (mosaic) aneuploidy be reliably detected in preimplantation embryos after multiple displacement amplification and single nucleotide polymorphism detection, independent of haplotyping and copy number detection, with a new method 'analysis of parental contribution for aneuploidy detection' or 'APCAD'? DesignThis method is based on the maternal contribution, a parameter that reflects the proportion of DNA that is of maternal origin for a given chromosome or chromosome segment. A maternal contribution deviating from 50% for autosomes is strongly indicative of a (mosaic) chromosomal anomaly. The method was optimized using cell mixtures with varying ratios of euploid and aneuploid (47,XY,+21) lymphocytes. Next, the maternal contribution was retrospectively measured for all chromosomes from 349 Karyomapping samples. ResultsRetrospective analysis showed a skewed maternal contribution (<36.4 or >63.6%) in 57 out of 59 autosome meiotic trisomies and all autosome monosomies (n = 57), with values close to theoretical expectation. Thirty-two out of 7436 chromosomes, for which no anomalies had been observed with Karyomapping, showed a similarly skewed maternal contribution. ConclusionsAPCAD was used to measure the maternal contribution, which is an intuitive parameter independent of copy number detection. This method is useful for detecting copy number neutral anomalies and can confirm diagnosis of (mosaic) aneuploidy detected based on copy number. Mosaic and complete aneuploidy can be distinguished and the parent of origin for (mosaic) chromosome anomalies can be determined. Because of these benefits, the APCAD method has the potential to improve aneuploidy detection carried out by comprehensive preimplantation genetic testing methods.

Detection of a novel single nucleotide polymorphism in IGF2 gene with a negative impact on egg production and body weight in Japanese quail (Coturnix japonica)

BackgroundInsulin-like growth factor 2 (IGF2) is one of three hormones that share high structural similarity to insulin. It is involved in several insulin-like growth-regulating and mitogenic activities. This study was conducted to genotype the coding regions of the IGF2 gene in Japanese quail (Coturnix japonica) using PCR-SSCP-sequencing, and to assess the possible association of the polymorphism of these regions with the main egg production traits. A total of 240 female birds with an equal number of three Japanese quail populations (Brown or BR, Black or BL, and White or WT) were included in this study. ResultsAll the genotyped regions exerted no heterogeneity in their sequences with one exception detected in the exon 2. In this locus, a novel single nucleotide polymorphism (SNP) was detected in which “A” was substituted with “G” at 81 position with a silent effect (p.F79=SNP) on IGF2 protein. Association analyses indicated a significant (P < 0.05) relation of this SNP with egg number (EN) and bird weight (BW) in the analyzed populations, in which the birds with AG genotype had lower EN and BW than those with AA genotype. The p.F79=SNP was largely detected in the WT line than the other two lines. ConclusionThe detected p.F79=SNP has a highly negative effect on EN and BW in Japanese quail. Thus, the implementation of the variations of the IGF2 gene can be a useful marker in the marker-assisted selection of Japanese quail. This is the first report to describe IGF2 gene variations in Japanese quail, which strongly suggests raising the birds from the BR line with AA genotype when breeders desire to raise Japanese quail for large-scale egg production.

Solid-Phase Primer Elongation Using Biotinylated dNTPs for the Detection of a Single Nucleotide Polymorphism from a Fingerprick Blood Sample.

Isothermal recombinase polymerase amplification-based solid-phase primer extension is used for the optical detection of a hypertrophic cardiomyopathy associated single nucleotide polymorphism (SNP) in a fingerprick blood sample. The assay exploits four thiolated primers which have the same sequences with the exception of the 3′-terminal base. Target DNA containing the SNP site hybridizes to all four of the immobilized probes, with primer extension only taking place from the primer containing the terminal base that is complementary to the SNP under interrogation. Biotinylated deoxynucleotide triphosphates are used in the primer extension, allowing postextension addition of streptavidin-poly-horseradish peroxidase to bind to the incorporated biotinylated dNTPs. The signal generated following substrate addition can then be measured optically. The percentage of biotinylated dNTPs and the duration of primer extension is optimized and the system applied to the identification of a SNP in a fingerprick blood sample. A methodology of thermal lysis using a 1 in 5 dilution of the fingerprick blood sample prior to application of 95 °C for 30 s is used to extract genomic DNA, which is directly used as a template for solid-phase primer extension on microtiter plates, followed by optical detection. The SNP in the fingerprick sample was identified and its identity corroborated using ion torrent next generation sequencing. Ongoing work is focused on extension to the multiplexed detection of SNPs in fingerprick and other biological samples.

Microfluidic Biosensor for Rapid Nucleic Acid Quantitation Based on Hyperspectral Interferometric Amplicon-Complex Analysis.

Nucleic acid detection plays a vital role in both biomedical research and clinical medicine. The temperature circulation changes of the widely used polymerase chain reaction technique are time-consuming and technically challenging for system development. Recombinase polymerase amplification (RPA) is an isothermal method for rapid nucleic acid detection. However, current RPA amplicon detection methods are complicated and expensive and easily generate false positives, restricting the promotion of RPA techniques. In this work, a hyperspectral interferometric amplicon-complex quantitation method is presented, combined with asymmetric dipole complex strategy optical scattering analysis. GelRed dye was utilized to form amplicon-complex particles, and the Fourier domain spectrum computation contributed to complex scattering quantitation. With this method, a supporting microfluidic chip and automatic system were developed to achieve integrated, rapid, quantitative, and miniscule nucleic acid detection. The Plasmodium falciparumdhfr gene was utilized as an example for targeted nucleic acid quantitation and single nucleotide polymorphism detection. The total reaction time was decreased to merely 20 min, and the limit of detection was only 3.17 ng/μL. The minimum measurable concentration of target was 1.68 copies/μL, 31.67 times more sensitive than turbidity detection, and the single reaction chamber was only 9.33 μL. No scattering increase occurred for template-free control, and thus, false positives caused by primer dimers and nonspecific products could be avoided. The experimental results prove that the provided method and system can detect single-base mutations in the dhfr gene and is a reasonable technique for rapid, automatic, and low-cost nucleic acid detection.

Development of a Single Quantum Dot-Mediated FRET Nanosensor for Sensitive Detection of Single-Nucleotide Polymorphism in Cancer Cells.

Single-nucleotide polymorphisms (SNPs) are important hallmarks of human diseases. Herein, we develop a single quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) nanosensor with the integration of multiple primer generation rolling circle amplification (MPG-RCA) for sensitive detection of SNPs in cancer cells. This assay involves only a linear padlock probe for MPG-RCA. The presence of a mutant target facilitates the circularization of linear padlock probes to initiate RCA, producing three short single-stranded DNAs (ssDNAs) with the assistance of nicking endonuclease. The resulting ssDNAs can function as primers to induce cyclic MPG-RCA, resulting in the exponential amplification and generation of large numbers of linker probes. The linker probes can subsequently hybridize with the Cy5-labeled reporter probes and the biotinylated capture probes to obtain the sandwich hybrids. The assembly of these sandwich hybrids on the 605 nm-emission quantum dot (605QD) generates the 605QD-oligonucleotide-Cy5 nanostructures, resulting in efficient FRET from the 605QD to Cy5. This nanosensor is free from both the complicated probe design and the exogenous primers and has distinct advantages of high amplification efficiency, zero background signal, good specificity, and high sensitivity. It can detect SNPs with a large dynamic range of 8 orders of magnitude and a detection limit of 5.41 × 10-20 M. Moreover, this nanosensor can accurately distinguish as low as 0.001% mutation level from the mixtures, which cannot be achieved by previously reported methods. Furthermore, it can discriminate cancer cells from normal cells and even quantify SNP at the single-cell level.

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection.

Background: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. Methods: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel B.1.617 lineages. Results: For the multiplex-test (SCOV2-617VOC-UCT), the analytic lower limit of detection was determined as 182 IU/mL for L452R, 144 IU/mL for P681R, and 79 IU/mL for E484Q. A total of 233 clinical samples were tested with the assay, including various on-target and off-target sequences. All SNPs (179/179 positive) were correctly identified as determined by SARS-CoV-2 whole genome sequencing. Conclusion: The recurrence of SNP locations and flexibility of methodology presented in this study allows for rapid adaptation to current and future variants. Furthermore, the ability to multiplex various SNP-assays into screening panels improves speed and efficiency for variant testing. We show 100% concordance with whole genome sequencing for a B.1.617.2 screening assay on the cobas6800 high-throughput system.

Bio-P-07 - Evaluation of mir-146a and mir-155 plasma expression levels in patients with mycosis fungoides and detection of single nucleotide polymorphisms in their sequence

<h3>Introduction:</h3> Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphomas presents with a broad spectrum of clinical and histopathologic manifestations. Diagnosis is often delayed, especially at the early stages. Clinicopathologic correlation and integration of molecular data is essential for a timely diagnosis and treatment. The use of microRNAs as biomarkers in biopsies and/or biological fluids has gained great interest over the last years. The aim of the present study was to analyze the expression levels of microRNAs (mir)-146a and -155 in the plasma of MF patients and healthy volunteers and to detect the presence of single nucleotide polymorphisms (SNPs) in their genes. <h3>Patients and methods:</h3> The appropriate sample size for statistical power equal to 0.94 (1–β=94%) and significance level at 0.05 (α=5%) was determined by a pilot study, using G.Power 3.1.9.2. The optimal sample size was calculated at n=82, with a ratio of MF patients/healthy individuals equal to 1:1. All samples were derived from consenting individuals. The mirs' plasma expression was evaluated with real-time quantitative reverse transcription PCR, and the relative quantity was calculated by the 2^–ΔCt method using cel-mir-39 as reference gene. In a subset of patients, the promoter region and/or the pre-microRNA genomic region of these mirs were scanned for the presence of SNPs in DNA extracted from white blood cells, by Sanger sequencing. Statistical analysis was performed with the statistical package IBM SPSS 25. <h3>Results:</h3> Plasma levels of mir-146a and mir-155 were significantly higher in patients with MF as compared to healthy volunteers (p=0.001 and p=0.028 respectively). Moreover, plasma levels were higher in patients with early MF vs healthy individuals (p=0.001 and p<0.001, for mir-146a and -155, respectively). Patients with advanced MF demonstrated higher expression levels of mir-146a and -155, when compared to early MF patients (p=0.009 and p=0.002). A positive correlation was detected between plasma levels of the two mirs in patients' samples (p<0.001). No correlation was detected between the expression of either mir and age/gender of the patients. The genotypes' frequencies for rs2910164 (CC/CG/GG) were 0.05/0.45/0.55 for MF patients and 0.1/0.54/0.36 for controls. The genotypes' frequencies for rs767649 (TT/AT/AA) were 0.76/0.18/0.06 for MF patients and 0.9/0.1/0 for controls. There was no significand difference in the genotype patterns between patients and controls for either SNP. No SNPs were detected in the pre-mir-155 sequence. <h3>Discussion:</h3> The presence of SNPs regulating mirs' function may need to be evaluated in larger cohorts, while the detection of increased mir-146a and mir-155 plasma levels in MF patients is an important finding in the attempt to establish putative non-invasive biomarkers for prompt diagnosis, prognosis, and adequate therapy of these patients.

Inclusion of double helix structural oligonucleotide (STexS) results in an enhance of SNP specificity in PCR

Genetic mutations such as single nucleotide polymorphisms (SNP) are known as one of the most common forms which related to various genetic disorders and cancers. Among of the methods developed for efficient detection of such SNP, polymerase chain reaction (PCR) methods are widely used worldwide for its cost and viable advantages. However, the technique to discriminate small amounts of SNP mixed in abundant normal DNA is incomplete due to intrinsic technical problems of PCR such as amplification occurring even in 3’mismatched cases because of high enzyme activity of DNA polymerases. To overcome the issue, specifically designed PCR platform, STexS (SNP typing with excellent specificity) using double stranded oligonucleotides was implemented as a means to emphasize the amplification of SNP templates by decreasing unwanted amplification of 3’mismatched DNA copies. In this study, the results indicate several EGFR mutations were easily detected specifically utilizing the STexS platform. Further trials show the novel method works effectively to discriminate mutations in not only general allele specific (AS)-PCRs, but also amplification refractory mutation system (ARMS)-PCR. The STexS platform will give aid in PCRs targeting potential SNPs or genetically mutated biomarkers in human clinical samples.

Monitoring the SARS-CoV-2 pandemic: screening algorithm with single nucleotide polymorphism detection for the rapid identification of established and emerging variants

ObjectivesTo evaluate a testing algorithm for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that includes the use of PCR-based targeted single nucleotide polymorphism (SNP) detection assays preceded by a multiplex PCR sensitive to S-Gene Target Failure (SGTF). MethodsPCR SNP assays targeting SARS-CoV-2 S-gene mutations ΔH69–V70, L452R, E484K, N501Y, H655Y and P681R using melting curve analysis were performed on 567 samples in which SARS-CoV-2 viral RNA was detected by a multiplex PCR. Viral whole-genome sequencing (WGS) was performed to confirm the presence of SNPs and to identify the Pangolin lineage. Additionally, 1133 SARS-CoV-2 positive samples with SGTF were further assessed by WGS to determine the presence of ΔH69–V70. ResultsThe N501Y-specific assay (n = 567) had an overall percentage agreement (OPA) of 98.5%. The ΔH69-V70-specific (n = 178) and E484K-specific (n = 401) assays had OPA of 96.6% and 99.7%, respectively. Assessment of H655Y (n = 139) yielded a 100.0% concordance when applied in the proposed algorithm. The L452R-specific (n = 67) and P681R-specific (n = 62) assays had an OPA of 98.2% and 98.1%, respectively. The proposed algorithm identified six variants of concern/interest (VOC/VOI)—Alpha (n = 149), Beta (n = 65), Gamma (n = 86), Delta (n = 49), Eta (n = 6), Kappa (n = 6)—and 205 non-VOC/VOI strains—including the variants under monitoring B.1.214.2 (n = 43) and B.1.1.318 (n = 18) and Epsilon (n = 1). An excellent concordance was observed for the identification of all SARS-CoV-2 lineages evaluated. ConclusionsWe present a flexible testing algorithm for the rapid detection of current and emerging SARS-CoV-2 VOC/VOIs, which can be easily adapted based on the local endemicity of specific variants.

The role of BDNF exon I region methylation in the treatment of depression with sertraline and its clinical diagnostic value.

BackgroundBrain‐derived neurotrophic factor (BDNF) is considered to be one of the best candidate genes for depression. However, whether sertraline treatment affects the methylation level of this gene remains unknown.MethodsFifty‐three patients with depression and 51 healthy controls were included in the study. The methylation level of BDNF exon I was determined in blood samples from these subjects. The Hamilton Depression Scale was used to evaluate the depression status of patients. Single nucleotide polymorphism detection was used for genotyping, and a receiver operating characteristic (ROC) curve was used to evaluate the predictive value of the methylation level of this locus in patients with depression.ResultsThere was a significant difference in the methylation level of BDNF exon I between the control and depression groups. No effect of sertraline monotherapy on BDNF methylation was found in subjects with depression. Moreover, no interaction was found between BDNF genotype and the per cent methylation of BDNF exon I. However, methylation at this site was positively correlated with diurnal variation and retardation scores. Blood homocysteine concentrations were significantly reduced by sertraline treatment. No influence of genotype on serum BDNF concentration was found in subjects with depression. The ROC curve showed that methylation of BDNF exon I may be used to distinguish patients from healthy people, to a certain extent.ConclusionMethylation of BDNF exon I may be used as a biomarker of depression and may be a therapeutic target for previously untreated depression.

SNP Development in Penaeus vannamei via Next-Generation Sequencing and DNA Pool Sequencing

Next-generation sequencing and pool sequencing have been widely used in SNP (single-nucleotide polymorphism) detection and population genetics research; however, there are few reports on SNPs related to the growth of Penaeus vannamei. The purpose of this study was to call SNPs from rapid-growing (RG) and slow-growing (SG) individuals’ transcriptomes and use DNA pool sequencing to assess the reliability of SNPs. Two parameters were applied to detect SNPs. One parameter was the p-values generated using Fisher’s exact test, which were used to calculate the significance of allele frequency differences between RG and SG. The other one was the AFI (minor allele frequency imbalance), which was defined to highlight the fold changes in MAF (minor allele frequency) values between RG and SG. There were 216,015 hypothetical SNPs, which were obtained based on the transcriptome data. Finally, 104 high-quality SNPs and 96,819 low-quality SNPs were predicted. Then, 18 high-quality SNPs and 17 low-quality SNPs were selected to assess the reliability of the detection process. Here, 72.22% (13/18) accuracy was achieved for high-quality SNPs, while only 52.94% (9/17) accuracy was achieved for low-quality SNPs. These SNPs enrich the data for population genetics studies of P. vannamei and may play a role in the development of SNP markers for future breeding studies.

OVarFlow: a resource optimized GATK\xa04 based Open source Variant calling workFlow

BackgroundThe advent of next generation sequencing has opened new avenues for basic and applied research. One application is the discovery of sequence variants causative of a phenotypic trait or a disease pathology. The computational task of detecting and annotating sequence differences of a target dataset between a reference genome is known as "variant calling". Typically, this task is computationally involved, often combining a complex chain of linked software tools. A major player in this field is the Genome Analysis Toolkit (GATK). The "GATK Best Practices" is a commonly referred recipe for variant calling. However, current computational recommendations on variant calling predominantly focus on human sequencing data and ignore ever-changing demands of high-throughput sequencing developments. Furthermore, frequent updates to such recommendations are counterintuitive to the goal of offering a standard workflow and hamper reproducibility over time.ResultsA workflow for automated detection of single nucleotide polymorphisms and insertion-deletions offers a wide range of applications in sequence annotation of model and non-model organisms. The introduced workflow builds on the GATK Best Practices, while enabling reproducibility over time and offering an open, generalized computational architecture. The workflow achieves parallelized data evaluation and maximizes performance of individual computational tasks. Optimized Java garbage collection and heap size settings for the GATK applications SortSam, MarkDuplicates, HaplotypeCaller, and GatherVcfs effectively cut the overall analysis time in half.ConclusionsThe demand for variant calling, efficient computational processing, and standardized workflows is growing. The Open source Variant calling workFlow (OVarFlow) offers automation and reproducibility for a computationally optimized variant calling task. By reducing usage of computational resources, the workflow removes prior existing entry barriers to the variant calling field and enables standardized variant calling.

O-089 A Genome Wide Association Study in men with unexplained infertility identifies nine SNPs at the FSHB locus to be associated with Follicle Stimulating Hormone level

Abstract Study question Which single nucleotide polymorphisms (SNPs) are associated with Follicle stimulating hormone (FSH) levels in men with unexplained infertility and can affect FSH action and spermatogenesis? Summary answer We identified a genomic region at chromosome 11p.14.1, including nine SNPs, that are significantly associated with FSH levels in men with unexplained infertility. What is known already FSH action is essential for the initiation and maintenance of human spermatogenesis. One well-studied SNP, FSHB c.-211G&amp;gt;T (rs10835638), is associated with FSHB mRNA transcription and directly affects FSH serum levels, testicular volume and spermatogenesis. Carriers of a T-allele in this promoter are diagnosed with functional secondary hypogonadism with isolated FSH deficiency. Other genetic variants, for example at the FSHR have been shown to slightly modulate FSH action, however the clinical impact in these variants seems to be low. The so far identified FSH-associated SNPs revealed an impact of up to 2.3 % on FSH serum level variance. Study design, size, duration A Genome wide association study (GWAS) was performed on a clinically well characterized cohort of 742 men with unexplained infertility (discovery study). Of the nine identified SNPs, validation was performed for rs11031005 and the already described rs10835638 in an independent cohort of 1123 men with unexplained infertility (validation study). Participants/materials, setting, methods Patients were retrospectively selected from our CeRA database Androbase® applying strict selection criteria; DNA was isolated from stored EDTA-blood samples. Informative genetic variants were identified using Illumina PsychArray v1.3. Illumina®GenomeStudio v2.0, PLINK v1.90 and R 3.6.3 were used to perform quantitative association analysis based on normalized FSH values. The validation study was performed using TaqMan PCR for SNP detection and R 3.6.3 for quantitative association to analyze the impact of each SNP on FSH level. Main results and the role of chance Imputation of the GWAS data revealed 94 SNPs with suggestive significance (p &amp;lt; 8.56e-06) and nine SNPs (including rs10835638) with genome-wide significance (p &amp;lt; 4.28e-07). The nine SNPs are all located at the FSHB locus on Chromosome 11p.14.1 and are in high linkage disequilibrium (LD). The validation study of 1123 patients with unexplained infertility for the SNPs rs11003005 and rs10835638 revealed a significant association with FSH (p = 4.71e-06 and p = 5.55e-07) and FSH/LH ratio (p = 2.08e-12 and p = 6.4e-12).The nine significant SNPs accounted for 3.60 –4.65 % variance in FSH serum level each in the entire discovery cohort. In an oligozoospermic subgroup (n = 249) this effect was increased to 4.89 – 6.95 %. This the first GWAS in men with unexplained infertility. This study shows that not one single SNP, but rather a genomic region has an impact on FSH serum level in men with unexplained male infertility. This effect is even more pronounced in the more severe phenotype of oligozoospermic men. Limitations, reasons for caution The study is restricted to men with unexplained infertility, which might cause a selection bias. Validation and functional evaluation of the eight newly identified SNPs in independent cohorts would emphasize the results more. The sample size of 742 limits detection of loci with smaller effect on FSH levels. Wider implications of the findings The determination of one of the nine SNPs can improve diagnostic precision in identifying men with secondary functional hypogonadism with isolated FSH deficiency. An oligozoospermic subgroup of these men would putatively benefit from FSH treatment and has to be proven in randomized controlled trials. Trial registration number German Research Foundation CRU326

Association of milk production traits with genetic variants in exon 5 and intron 3 of bovine growth hormone (bGH) gene in Sahiwal cattle

The main objective of this study was to determine the association of production traits with genetic variants in exon 5 and intron 3 of bovine growth hormone (bGH) gene in Sahiwal cattle. The analyses were based on the detection of single nucleotide polymorphisms (SNPs) in GH-AluI (exon 5) and GH-MspI (intron 3) using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The frequency of AluI (L/V) and MspI (T/C) alleles were 86.20/13.70 and 89.80/9.50, respectively. The distribution of the frequency of GH genotypes for LL, LV, and VV were 73.4, 25.5, and 1.0 and for TT, TC, and CC genotypes were 80.9, 17.7, and 13.0, respectively. Season of calving had none but period of calving had significant effect on the studied production traits. The phenotypic data was adjusted for non-genetic factors and regression analysis was done. There was detectable significant effect of the GH-AluI and GH-MspI on the analyzed production traits. The cows with LL genotype had significantly higher milk yield traits than the LV genotype. Similarly, the productive performance of the studied cows with TT genotype was also significantly higher than TC and CC genotypes. Results revealed that the Sahiwal cows with LL and TT genotypes of the bGH locus can be considered to be a favourable genotype for production traits, although these findings need to be confirmed by further research before SNPs can be used in a marker assisted selection program by the animal breeder.