A Practical and User-Friendly Method for the Selenium-Free One-Step Preparation of 1,2-Diketones and their Monoxime Analogs

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6+ or ura4+ genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.

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A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

Journal of Tropical Medicine ◽

10.1155/2017/7210849 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Nur Amalina Khazani ◽

Nik Zuraina Nik Mohd Noor ◽

Chan Yean Yean ◽

Habsah Hasan ◽

Siti Suraiya ◽

...

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Cold Chain ◽

Molecular Diagnostic ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

One Step ◽

The Stability ◽

Tract Infections ◽

User Friendly

Klebsiella pneumoniaeandHaemophilus influenzaeare two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection ofK. pneumoniaeandH. influenzae.The multiplex PCR assay was designed to amplify thephpgene ofK. pneumoniae(202 bp) andp6gene ofH. influenzae(582 bp). In addition, the specific primer to amplifyglmgene ofHelicobacter pylori(105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using theQ10method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and theQ10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection ofK. pneumoniaeandH. influenzaewas successfully developed in this study.

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SPEAQeasy: a scalable pipeline for expression analysis and quantification for R/bioconductor-powered RNA-seq analyses

BMC Bioinformatics ◽

10.1186/s12859-021-04142-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nicholas J. Eagles ◽

Emily E. Burke ◽

Jacob Leonard ◽

Brianna K. Barry ◽

Joshua M. Stolz ◽

...

Keyword(s):

Expression Analysis ◽

Computational Domain ◽

Rna Seq ◽

Entry Barrier ◽

One Step ◽

Differential Gene ◽

Main Input ◽

Set Up ◽

Downstream Analysis ◽

User Friendly

Abstract Background RNA sequencing (RNA-seq) is a common and widespread biological assay, and an increasing amount of data is generated with it. In practice, there are a large number of individual steps a researcher must perform before raw RNA-seq reads yield directly valuable information, such as differential gene expression data. Existing software tools are typically specialized, only performing one step–such as alignment of reads to a reference genome–of a larger workflow. The demand for a more comprehensive and reproducible workflow has led to the production of a number of publicly available RNA-seq pipelines. However, we have found that most require computational expertise to set up or share among several users, are not actively maintained, or lack features we have found to be important in our own analyses. Results In response to these concerns, we have developed a Scalable Pipeline for Expression Analysis and Quantification (SPEAQeasy), which is easy to install and share, and provides a bridge towards R/Bioconductor downstream analysis solutions. SPEAQeasy is portable across computational frameworks (SGE, SLURM, local, docker integration) and different configuration files are provided (http://research.libd.org/SPEAQeasy/). Conclusions SPEAQeasy is user-friendly and lowers the computational-domain entry barrier for biologists and clinicians to RNA-seq data processing as the main input file is a table with sample names and their corresponding FASTQ files. The goal is to provide a flexible pipeline that is immediately usable by researchers, regardless of their technical background or computing environment.

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Kitchen Range Oven Enabled One-tube RT-LAMP for RNA Detection at Home - A Potential Solution for Large-scale Screening of COVID-19

10.31224/osf.io/ed85s ◽

2020 ◽

Cited By ~ 1

Author(s):

Yu Lei

Keyword(s):

Large Scale ◽

Lamp Reaction ◽

Sample Collection ◽

Rna Detection ◽

Closed Tube ◽

One Step ◽

User Friendly ◽

Target Rna ◽

Large Scale Screening ◽

At Home

Rapid, low-cost, and user-friendly molecular diagnostic methods are prerequisite to address the outbreaks of infectious diseases. Especially during the outbreak of COVID-19 pandemic, there is an urgent need to build the global testing capacity up to 100-fold above what is achievable with current standard approaches. However, current gold standard methods such as RT-PCR and isothermal PCR (e.g., RT-LAMP), which are routinely conducted in laboratories, suffer from limit capacity due to the requirement of special equipment, multiple sample handling steps, and/or the need of skilled personnel. In this study, a kitchen Range Oven enabled RT-LAMP was conducted at a residential home without sacrificing the performance in RNA detection. In addition, one-step, closed-tube RT-LAMP for the detection of target RNA in the oven was accomplished by pre-loading sample collection solution and RT-LAMP reaction reagents into the bottom and cap cavity of PCR tube, respectively. After the addition of target RNA into sample collection solution through either swab swirling or direct pipetting, a flip-and-swing enabled mixing of sample solution and RT-LAMP reaction reagents was conducted first, followed by RT-LAMP detection at a constant temperature in pre-heated oven. The RNA positive sample can be obviously differentiated from the RNA negative sample through both naked-eye based turbidity detection and fluorescence detection under UV light. In conjunction with the user-friendly one-step, closed-tube concept, this study indicates that it is feasible to run RT-LAMP at home using oven with minimum involvement of end-users, thus offering an excellent molecular detection platform, which has the potential to boost the detection capacity for any nucleic acid target. It could be a potential solution for large-scale screening of COVID-19 in short time as the tests can be conducted by residents at home, without the need of well-trained health caregivers and capacity limitation.

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SPEAQeasy: a Scalable Pipeline for Expression Analysis and Quantification for R/Bioconductor-powered RNA-seq analyses

10.1101/2020.12.11.386789 ◽

2020 ◽

Author(s):

Nicholas J. Eagles ◽

Emily E. Burke ◽

Jabob Leonard ◽

Brianna K. Barry ◽

Joshua M. Stolz ◽

...

Keyword(s):

Expression Analysis ◽

Computational Domain ◽

Rna Seq ◽

Entry Barrier ◽

One Step ◽

Differential Gene ◽

Main Input ◽

Set Up ◽

Downstream Analysis ◽

User Friendly

RNA sequencing (RNA-seq) is a common and widespread biological assay, and an increasing amount of data is generated with it. In practice, there are a large number of individual steps a researcher must perform before raw RNA-seq reads yield directly valuable information, such as differential gene expression data. Existing software tools are typically specialized, only performing one step-- such as alignment of reads to a reference genome-- of a larger workflow. The demand for a more comprehensive and reproducible workflow has led to the production of a number of publicly available RNA-seq pipelines. However, we have found that most require computational expertise to set up or share among several users, are not actively maintained, or lack features we have found to be important in our own analyses. In response to these concerns, we have developed a Scalable Pipeline for Expression Analysis and Quantification (SPEAQeasy), which is easy to install and share, and provides a bridge towards R/Bioconductor downstream analysis solutions. SPEAQeasy is user-friendly and lowers the computational-domain entry barrier for biologists and clinicians to RNA-seq data processing as the main input file is a table with sample names and their corresponding FASTQ files. SPEAQeasy is portable across computational frameworks (SGE, SLURM, local, docker integration) and different configuration files are provided.

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A 4-lineage statistical suite to evaluate the support of large-scale retrotransposon insertion data to reconstruct evolutionary trees

10.1101/2020.12.10.419374 ◽

2020 ◽

Author(s):

G Churakov ◽

A Kuritzin ◽

K Chukharev ◽

F Zhang ◽

F Wünnemann ◽

...

Keyword(s):

Large Scale ◽

Incomplete Lineage Sorting ◽

Evolutionary Trees ◽

Lineage Sorting ◽

High Throughput Data ◽

Genome Wide ◽

Tree Topologies ◽

One Step ◽

General Scientific ◽

User Friendly

AbstractRetrophylogenomics makes use of genome-wide retrotransposon presence/absence insertion patterns to resolve questions in phylogeny and population genetics. In the genomics era, evaluating high-throughput data requires the associated development of appropriately powerful statistical tools. The currently used KKSC 3-lineage statistical test for evaluating the significance of data is limited by the number of possible tree topologies it can assess in one step. To improve on this, we have now extended the analysis to simultaneously compare 4-lineages, which now enables us to evaluate ten distinct presence/absence insertion patterns for 26 possible tree topologies plus 129 trees with different incidences of hybridization. Moreover, the new tool includes statistics for multiple ancestral hybridizations, ancestral incomplete lineage sorting, bifurcation, and polytomy. The test is embedded in a user-friendly web R-application (http://retrogenomics.uni-muenster.de:3838/hammlet/) and is available for use by the general scientific community.

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One-step pipetting of barcoded planar microparticles into compact monolayer assembling chip for efficient readout of multiplexed immunoassay

10.1101/2022.01.03.474850 ◽

2022 ◽

Author(s):

Sangwook Bae ◽

Daewon Lee ◽

Hunjong Na ◽

Jiseong Jang ◽

Sunghoon Kwon

Keyword(s):

Capillary Flow ◽

Straightforward Manner ◽

Particle Assembly ◽

Research Level ◽

One Step ◽

Image Processing Algorithms ◽

Cost Efficient ◽

Multiplexed Immunoassay ◽

User Friendly ◽

Soft Elastomer

Barcoded planar microparticles have many qualities suitable for developing cost-efficient multiplexed immunoassays. But at the translational research level, there are a number of technical aspects yet remain to be addressed which includes robustness and efficiency of the assay readout process. Assay readout process involves automated barcode identification and signal intensity values from each planar microparticle. For this, each microparticle has to be correctly aligned for correct barcode readout while being, ideally, compactly assembled for maximum microparticle imaging efficiency. To simultaneously achieve such alignment and assembly of microparticles but in a straightforward manner, we designed a microfluidic microparticle assembling chip that only requires a single pipetting step. Our design utilizes capillary flow based guided particle assembly, which allows maximum microparticle-based immunoassay readout efficiency. With the aid of image processing algorithms, we obtained good multiplex immunoassay readout accuracy similar to conventional imaging platforms. Our approach is applicable to both soft elastomer materials (e.g. PDMS) and rigid materials (e.g. polystyrene), the latter of which is frequently used for injection molding based mass production. We anticipate our device could help developing facile and user-friendly platform technologies based on barcoded planar microparticles.

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Handheld, One-Step, and Rapid Electrochemical Biosensor Platform with Smartphone Interface

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.868.340 ◽

2017 ◽

Vol 868 ◽

pp. 340-344 ◽

Cited By ~ 1

Author(s):

Jin An Wang ◽

Zhong Liang Xia ◽

Yan Su ◽

Min Cong Lu ◽

Ying Wan ◽

...

Keyword(s):

Screen Printing ◽

Electrochemical Biosensor ◽

Point Of Care ◽

Detection Time ◽

Maximum Residue Limit ◽

Point Of Care Testing ◽

Step Detection ◽

Electrode Sensor ◽

One Step ◽

User Friendly

Organophosphorous pesticide (OP), one of the most commonly used pesticides, could have serious adverse effects on human health and the environment. As the maximum residue limit (MRL) of OPs in food and water is low, it is in great demand to develop integrated, portable, user-friendly devices for the rapid and sensitive detection of OP residue. This work aims to present a homemade handhold device and screen printing electrode (SPE) chip for rapid, sensitive, and one-step detection of OPs. A handhold device was designed and fabricated to connect with smartphone to readout the electrochemical signals. Taking advantage of a three-electrode sensor chip and the inhibiting reaction of acetylcholine esterase (AchE), Trichlorphon can be detected in a considerable low detection limit of 1 ng/mL. By using the as-fabricated device and chip, the detection time was only 5 min and each test costs less than $1. The platform will allow point-of-care testing (POCT) of OPs and other targets, which shows promise for healthy and environmental monitoring.

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A Practical and User-Friendly Method for the Selenium-Free One-Step Preparation of 1,2-Diketones and Their Monoxime Analogues.

ChemInform ◽

10.1002/chin.200517046 ◽

2005 ◽

Vol 36 (17) ◽

Author(s):

Georg Rueedi ◽

Matthias A. Oberli ◽

Matthias Nagel ◽

Christophe Weymuth ◽

Hans-Juergen Hansen

Keyword(s):

One Step ◽

User Friendly

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An Interactive Minicomputer Program for the Indexing and Simulation of Electron Diffraction Patterns

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092670 ◽

1976 ◽

Vol 34 ◽

pp. 542-543

Author(s):

R.P. Goehner ◽

W.T. Hatfield ◽

Prakash Rao

Keyword(s):

Electron Diffraction ◽

Real Time ◽

Pattern Analysis ◽

Flow Diagram ◽

Hard Copy ◽

Time Analysis ◽

Real Time Analysis ◽

Transmission Electron ◽

One Step ◽

Diffraction Patterns

Computer programs are now available in various laboratories for the indexing and simulation of transmission electron diffraction patterns. Although these programs address themselves to the solution of various aspects of the indexing and simulation process, the ultimate goal is to perform real time diffraction pattern analysis directly off of the imaging screen of the transmission electron microscope. The program to be described in this paper represents one step prior to real time analysis. It involves the combination of two programs, described in an earlier paper(l), into a single program for use on an interactive basis with a minicomputer. In our case, the minicomputer is an INTERDATA 70 equipped with a Tektronix 4010-1 graphical display terminal and hard copy unit.A simplified flow diagram of the combined program, written in Fortran IV, is shown in Figure 1. It consists of two programs INDEX and TEDP which index and simulate electron diffraction patterns respectively. The user has the option of choosing either the indexing or simulating aspects of the combined program.

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