Next-generation sequencing provides useful information about gene mutations, gene expression, epigenetic modification, microRNA expression, and copy number variations. More and more computing tools ...have been developed to analyze this large quantity of information. However, to test and find suitable analytical tools and integrate their results is tedious and challenging for users with little bioinformatics training. In the present study, we assembled the computing tools into a convenient toolkit to simplify the analysis and integration of data between bioinformatics tools.
The toolkit, GeneGazer, comprises of two parts: the first, named Gaze_Profiler, was designed for personalized molecular profiling from next-generation sequencing data of paired samples; the other, named Gaze_BioSigner, was designed for the discovery of disease-associated biosignatures from expressional and mutational profiles of a cohort study.
To demonstrate the capabilities of Gaze_Profiler, we analyzed a pair (colon cancer and adjacent normal tissues) of RNA-sequencing data from one patient downloaded from the Sequencing Read Archive database and used them to profile somatic mutations and digital gene expression. In this case, alterations in the RAS/RAF/MEK/ERK signaling pathway (activated by KRAS G13D mutation) and canonical WNT signaling pathway (activated by truncated APC) were identified; no EGFR mutation or overexpression was found. These data suggested a limited efficacy of cetuximab in the patient. To demonstrate the ability of Gazer_BioSigner, we analyzed gene-expression data from 192 cancer tissues downloaded from The Cancer Genome Atlas and found that the activation of cAMP/PKA signaling, OCT-3/4 and SRF were associated with colon cancer progression and could be potential therapeutic targets.
GeneGazer is a reliable and robust toolkit for the analysis of data from high-throughput platforms and has potential for clinical application and biomedical research.
▸ The detection of oligonucleotides in a range 0.5–500pM was demonstrated. ▸ Perfectly matched oligonucleotide at 10, 15 and 20-mer were measured. ▸ The detection limit of PSPWB on oligonucleotides ...hybridization is 0.5pM in PBS.
Detection of unlabeled oligonucleotides using surface plasmon resonance (SPR) is difficult because of the oligonucleotides’ relatively lower molecular weight compared with proteins. In this paper, we describe a method for detecting unlabeled oligonucleotides at low concentration using a paired surface plasma waves biosensor (PSPWB). The biosensor uses a sensor chip with an immobilized probe to detect a target oligonucleotide via sequence-specific hybridization. PSPWB measures the demodulated amplitude of the heterodyne signal in real time. In the meantime, the ratio of the amplitudes between the detected output signal and reference can reduce the excess noise from the laser intensity fluctuation. Also, the common-path propagation of p and s waves cancels the common phase noise induced by temperature variation. Thus, a high signal-to-noise ratio (SNR) of the heterodyne signal is detected. The sequence specificity of oligonucleotide hybridization ensures that the platform is precisely discriminating between target and non-target oligonucleotides. Under optimized experimental conditions, the detected heterodyne signal increases linearly with the logarithm of the concentration of target oligonucleotide over the range 0.5–500pM. The detection limit is 0.5pM in this experiment. In addition, the non-target oligonucleotide at concentrations of 10pM and 10nM generated signals only slightly higher than background, indicating the high selectivity and specificity of this method. Different length of perfectly matched oligonucleotide targets at 10-mer, 15-mer and 20-mer were identified at the concentration of 150pM.
5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ...ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.
Noonan syndrome (NS, OMIM 163950) is a relatively common autosomal dominant disorder and has significant phenotypic overlap with Costello Syndrome and cardio-facio-cutaneous syndrome. Molecular ...diagnosis is useful for differential diagnosis.
PTPN11 gene mutation is the most common mutation associated with NS and hence is a suitable target for molecular diagnostics.
High resolution melting (HRM) analysis was used for screening of
PTPN11 mutations. Eleven DNA samples with 10 known
PTPN11 mutations were used for HRM calibration. Said calibrations were then applied to mutation screening of a panel of 50 additional NS patients.
HRM analysis differentiated all of the 10 known mutations and identified 9 additional mutations from 10 patients in the blind study, which is in line with results obtained by sequencing.
HRM analysis is a rapid, reliable, and low-cost tool for detection of
PTPN11 genetic variants.
Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage
. The FA repair pathway protects against ...endogenous and exogenous carcinogenic aldehydes
. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas
(SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus
(HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.
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In‐stent restenosis (ISR) is a pathological phenomenon in response to vascular injury after balloon angioplasty with stent placement, which occurs in just about 20~30% cases. Because of ...the lack of ISR‐specific clinical biomarkers, the drug‐eluted stents (DES) were widely abused as preventive interventions at present. A growing number of evidences suggested that abnormal expression profiles of microRNAs (miRNAs) associated with development of numerous diseases. To date, no reports showed that any miRNA candidates have been used to distinguish the population with high‐risk for ISR. Thereby, the purpose of the present study was to identify potential candidates of miRNAs differentially expressed in ISR patients. Based on the correlation network of our preliminary data from the proteiomics analysis of animal disease model and a small‐size SNP array of human samples, the candidate miRNAs with highly potential to involve in ISR development were picked up by using the MetaCore™ software. Thirty participants, including the restenosis group and the non‐restenosis group, were recruited from the Taipei Medical University Hospital, Taiwan. Total miRNA was extracted from blood serum of each participant, and expression profiles of five candidate miRNAs (mir‐142‐5p, mir‐132‐3p, mir‐24‐2‐5p, mir‐424‐5p and mir‐15b‐5p) were analyzed by using SYBR Green‐based real‐time PCR. Our data mentioned that the expression levels of two mRNAs (mir‐424‐5p and mir‐15b‐5p) showed increasing trends but another mRNAs (mir‐142‐5p, mir‐132‐3p and mir‐24‐2‐5p) were decreased in the restenosis group.
Support or Funding Information
Taipei Medical University
Taipei Medical University Hospital
The sensing properties of Au-EGFET with body effect for DNA detection were first investigated in this study. The body effect means different substrate bias in CMOS circuit operation without common ...ground. With positive V BS of 1V, more shift of I D -V G curve could be a good index of DNA hybridization and concentration. It could be explained be the electric field induced counter ion effect. This measured technique can be used to enhance the sensing signal and improve the limit of detection (LOD).
