scholarly journals THE COMPARISON OF THREE REAL-TIME PCR KITS FOR SARS-COV-2 DIAGNOSIS REVEALS DISCREPANCIES ON THE IDENTIFICATION OF POSITIVE COVID-19 CASES AND DISPERSION ON THE VALUES OBTAINED FOR THE DETECTION OF SARS-COV-2 VARIANTS

2021 ◽  
Author(s):  
Alvaro Santibanez ◽  
Roberto Luraschi ◽  
Carlos Barrera-Avalos ◽  
Eva Vallejos-Vidal ◽  
Javiera Alarcon ◽  
...  
Keyword(s):  
Real Time ◽  
Rna Virus ◽  
Nasopharyngeal Swab ◽  
World Population ◽  
Single Nucleotide Variants ◽  
Reaction Conditions ◽  
Single Nucleotide ◽  
Total Rna ◽  
Challenge And Threat ◽  
And Control

The COVID-19 pandemic has generated a huge challenge and threat to public health throughout the world population. Reverse transcription associated with real-time Polymerase Chain Reaction (RT-qPCR) has been the gold-standard molecular tool for diagnosis and detection of the SARS-CoV-2. Currently, it is used as the main strategy for testing, traceability, and control of positive cases For this reason, the on-top high demand for reagents has produced stock-out on several occasions and the only alternative to keep population diagnosis has been the use of different RT-qPCR kits. Therefore, we evaluate the performance of three of the commercial RT-qPCR kits currently in use for SARS-CoV-2 diagnosis in Chile, consisting in: TaqMan 2019-nCoV Assay Kit v1 (Thermo). Real-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2 (BGI), and LightCycler Multiplex RNA Virus Master (Roche). Results of quantification cycle (Cq) and relative fluorescence units (RFU) obtained from their RT-qPCR reactions revealed important discrepancies on the total RNA required for the identification of SARS-CoV-2 genes and diagnosis. Marked differences between kits in samples with 30>Cq value< 34 was observed. Samples with positive diagnoses for Covid-19 using the Thermo Fisher kit had different results when the same samples were evaluated with Roche and BGI kits. The displacement on the Cq value for SARS-CoV-2 identification between the three different RT-qPCR kits was also evident when the presence of single nucleotide variants was evaluated in the context of genomic surveillance. Taken together, this study emphasizes the special care adjusting RT-qPCR reaction conditions of the different kits must be taken by all the laboratories before carrying out the detection of SARS-CoV-2 genes from total RNA nasopharyngeal swab (NPS) samples.

scholarly journals Association between HOX Transcript Antisense RNA Single-Nucleotide Variants and Recurrent Implantation Failure

2021 ◽  
Vol 22 (6) ◽  
pp. 3021
Author(s):  
Jeong Yong Lee ◽  
Eun Hee Ahn ◽  
Hyeon Woo Park ◽  
Ji Hyang Kim ◽  
Young Ran Kim ◽  
...  
Keyword(s):  
Antisense Rna ◽  
Coagulation Factors ◽  
Healthy Controls ◽  
Implantation Failure ◽  
Single Nucleotide Variants ◽  
Recurrent Implantation Failure ◽  
Single Nucleotide ◽  
Vitro Fertilization ◽  
And Control

Recurrent implantation failure (RIF) refers to the occurrence of more than two failed in vitro fertilization–embryo transfers (IVF-ETs) in the same individual. RIF can occur for many reasons, including embryo characteristics, immunological factors, and coagulation factors. Genetics can also contribute to RIF, with some single-nucleotide variants (SNVs) reported to be associated with RIF occurrence. We examined SNVs in a long non-coding RNA, homeobox (HOX) transcript antisense RNA (HOTAIR), which is known to affect cancer development. HOTAIR regulates epigenetic outcomes through histone modifications and chromatin remodeling. We recruited 155 female RIF patients and 330 healthy controls, and genotyped HOTAIR SNVs, including rs4759314, rs920778, rs7958904, and rs1899663, in all participants. Differences in these SNVs were compared between the patient and control groups. We identified significant differences in the occurrence of heterozygous genotypes and the dominant expression model for the rs1899663 and rs7958904 SNVs between RIF patients and control subjects. These HOTAIR variants were associated with serum hemoglobin (Hgb), luteinizing hormone (LH), total cholesterol (T. chol), and blood urea nitrogen (BUN) levels, as assessed by analysis of variance (ANOVA). We analyzed the four HOTAIR SNVs and found significant differences in haplotype patterns between RIF patients and healthy controls. The results of this study showed that HOTAIR is not only associated with the development of cancer but also with pregnancy-associated diseases. This study represents the first report showing that HOTAIR is correlated with RIF.

scholarly journals A short plus long-amplicon based sequencing approach improves genomic coverage and variant detection in the SARS-CoV-2 genome

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261014
Author(s):  
Carlos Arana ◽  
Chaoying Liang ◽  
Matthew Brock ◽  
Bo Zhang ◽  
Jinchun Zhou ◽  
...  
Keyword(s):  
Virus Genome ◽  
Positive Control ◽  
Nasopharyngeal Swab ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Spike Gene ◽  
Synonymous Mutations ◽  
Variant Detection ◽  
Variant Analysis ◽  
New Mutations

High viral transmission in the COVID-19 pandemic has enabled SARS‐CoV‐2 to acquire new mutations that may impact genome sequencing methods. The ARTIC.v3 primer pool that amplifies short amplicons in a multiplex-PCR reaction is one of the most widely used methods for sequencing the SARS-CoV-2 genome. We observed that some genomic intervals are poorly captured with ARTIC primers. To improve the genomic coverage and variant detection across these intervals, we designed long amplicon primers and evaluated the performance of a short (ARTIC) plus long amplicon (MRL) sequencing approach. Sequencing assays were optimized on VR-1986D-ATCC RNA followed by sequencing of nasopharyngeal swab specimens from fifteen COVID-19 positive patients. ARTIC data covered 94.47% of the virus genome fraction in the positive control and patient samples. Variant analysis in the ARTIC data detected 217 mutations, including 209 single nucleotide variants (SNVs) and eight insertions & deletions. On the other hand, long-amplicon data detected 156 mutations, of which 80% were concordant with ARTIC data. Combined analysis of ARTIC + MRL data improved the genomic coverage to 97.03% and identified 214 high confidence mutations. The combined final set of 214 mutations included 203 SNVs, 8 deletions and 3 insertions. Analysis showed 26 SARS-CoV-2 lineage defining mutations including 4 known variants of concern K417N, E484K, N501Y, P618H in spike gene. Hybrid analysis identified 7 nonsynonymous and 5 synonymous mutations across the genome that were either ambiguous or not called in ARTIC data. For example, G172V mutation in the ORF3a protein and A2A mutation in Membrane protein were missed by the ARTIC assay. Thus, we show that while the short amplicon (ARTIC) assay provides good genomic coverage with high throughput, complementation of poorly captured intervals with long amplicon data can significantly improve SARS-CoV-2 genomic coverage and variant detection.

scholarly journals Unexpected CRISPR off-target mutation pattern in vivo are not typically germline-like

2017 ◽  
Author(s):  
Zhiting Wei ◽  
Funan He ◽  
Guohui Chuai ◽  
Hanhui Ma ◽  
Zhixi Su ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Cas9 Nuclease ◽  
Mutation Pattern ◽  
And Control ◽  
Selection Of

To the EditorSchaefer et al.1 (referred to as Study_1) recently presented the provocative conclusion that CRISPR-Cas9 nuclease can induce many unexpected off-target mutations across the genome that arise from the sites with poor homology to the gRNA. As Wilson et al.2 pointed out, however, the selection of a co-housed mouse as the control is insufficient to attribute the observed mutation differences between the CRISPR-treated mice and control mice. Therefore, the causes of these mutations need to be further investigated. In 2015, Iyer et al.3 (referred to as Study_2) used Cas9 and a pair of sgRNAs to mutate the Ar gene in vivo and off-target mutations were investigated by comparison the control mice and the offspring of the modified mice. After analyzing the whole genome sequencing (WGS) of the offspring and the control mice, they claimed that off-target mutations are rare from CRISPR-Cas9 engineering. Notably, their study only focused on indel off-target mutations. We re-analyzed the WGS data of these two studies and detected both single nucleotide variants (SNVs) and indel mutations.

scholarly journals Identification and Characterization of Splicing Defects by Single-Molecule Real-Time Sequencing Technology (PacBio)

2020 ◽  
Vol 7 (4) ◽  
pp. 477-481
Author(s):  
Marco Savarese ◽  
Talha Qureshi ◽  
Annalaura Torella ◽  
Pia Laine ◽  
Teresa Giugliano ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Genetic Diseases ◽  
Disease Genes ◽  
Test Results ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Dna Test ◽  
Splicing Defects

Although DNA-sequencing is the most effective procedure to achieve a molecular diagnosis in genetic diseases, complementary RNA analyses are often required. Reverse-Transcription polymerase chain reaction (RT-PCR) is still a valuable option when the clinical phenotype and/or available DNA-test results address the diagnosis toward a gene of interest or when the splicing effect of a single variant needs to be assessed. We use Single-Molecule Real-Time sequencing to detect and characterize splicing defects and single nucleotide variants in well-known disease genes (DMD, NF1, TTN). After proper optimization, the procedure could be used in the diagnostic setting, simplifying the workflow of cDNA analysis.

Microstructural investigation of surface roughness in MBE-grown AlAs

1995 ◽  
Vol 53 ◽  
pp. 454-455
Author(s):  
R. Rajesh ◽  
R. Droopad ◽  
C. H. Kuo ◽  
R. W. Carpenter ◽  
G. N. Maracas
Keyword(s):  
Thin Film ◽  
Real Time ◽  
Growth Control ◽  
Native Oxide ◽  
Effusion Cell ◽  
Surface Oxide Layer ◽  
Microstructural Investigation ◽  
Mbe Growth ◽  
Film Substrate ◽  
And Control

Knowledge of material pseudodielectric functions at MBE growth temperatures is essential for achieving in-situ, real time growth control. This allows us to accurately monitor and control thicknesses of the layers during growth. Undesired effusion cell temperature fluctuations during growth can thus be compensated for in real-time by spectroscopic ellipsometry. The accuracy in determining pseudodielectric functions is increased if one does not require applying a structure model to correct for the presence of an unknown surface layer such as a native oxide. Performing these measurements in an MBE reactor on as-grown material gives us this advantage. Thus, a simple three phase model (vacuum/thin film/substrate) can be used to obtain thin film data without uncertainties arising from a surface oxide layer of unknown composition and temperature dependence.In this study, we obtain the pseudodielectric functions of MBE-grown AlAs from growth temperature (650°C) to room temperature (30°C). The profile of the wavelength-dependent function from the ellipsometry data indicated a rough surface after growth of 0.5 μm of AlAs at a substrate temperature of 600°C, which is typical for MBE-growth of GaAs.

Adaptive Signal Analysis and Interpretation for Real-time Intelligent Patient Monitoring

1994 ◽  
Vol 33 (01) ◽  
pp. 60-63 ◽  
Author(s):  
E. J. Manders ◽  
D. P. Lindstrom ◽  
B. M. Dawant
Keyword(s):  
Computer Architecture ◽  
Real Time ◽  
Data Abstraction ◽  
Monitoring Strategy ◽  
Intelligent Monitoring ◽  
Patient Status ◽  
On Line ◽  
Main Components ◽  
And Control ◽  
Adaptive Signal

Abstract:On-line intelligent monitoring, diagnosis, and control of dynamic systems such as patients in intensive care units necessitates the context-dependent acquisition, processing, analysis, and interpretation of large amounts of possibly noisy and incomplete data. The dynamic nature of the process also requires a continuous evaluation and adaptation of the monitoring strategy to respond to changes both in the monitored patient and in the monitoring equipment. Moreover, real-time constraints may imply data losses, the importance of which has to be minimized. This paper presents a computer architecture designed to accomplish these tasks. Its main components are a model and a data abstraction module. The model provides the system with a monitoring context related to the patient status. The data abstraction module relies on that information to adapt the monitoring strategy and provide the model with the necessary information. This paper focuses on the data abstraction module and its interaction with the model.

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