scholarly journals MultiEditR: An easy validation method for detecting and quantifying RNA editing from Sanger sequencing

2019 ◽  
Author(s):  
Mitchell Kluesner ◽  
Annette Arnold ◽  
Taga Lerner ◽  
Rafail Nikolaos Tasakis ◽  
Sandra Wüst ◽  
...  
Keyword(s):  
Rna Editing ◽  
Sanger Sequencing ◽  
Tumour Progression ◽  
Cost Effective ◽  
Base Change ◽  
Rna Seq ◽  
Multiple Sources ◽  
Base Editing ◽  
Cost Effective Method ◽  
Apobec Family

ABSTRACTRNA editing is the base change that results from RNA deamination by two predominant classes of deaminases; the APOBEC family and the ADAR family. Respectively, deamination of nucleobases by these enzymes are responsible for endogenous editing of cytosine to uracil (C-to-U) and adenosine to inosine (A-to-I). RNA editing is known to play an essential role both in maintaining normal cellular function, as well as altered cellular physiology during oncogenesis and tumour progression. Analysis of RNA editing in these important processes, largely relies on RNA-seq technology for the detection and quantification of RNA editing sites. Despite the power of these technologies, multiple sources of error in detecting and measuring base editing still exist, therefore additional validation and quantification of editing through Sanger sequencing is still required for confirmation of editing. Depending on the number of RNA editing sites that are of interest, this validation step can be both expensive and time-consuming. To address this need we developed the tool MultiEditR which provides a simple, and cost-effective method of detecting and quantifying RNA editing form Sanger sequencing. We expect that MultiEditR will foster further discoveries in this rapidly expanding field.

scholarly journals Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 283
Author(s):  
Eyal Seroussi
Keyword(s):  
Copy Number ◽  
Sanger Sequencing ◽  
Cytosine Methylation ◽  
Direct Sequencing ◽  
Information Source ◽  
Gene Copy Number ◽  
Cost Effective ◽  
Gene Copy ◽  
Base Editing ◽  
Recent Developments

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

scholarly journals High throughput sequencing of multiple amplicons for barcoding and integrative taxonomy

2016 ◽  
Author(s):  
Perrine Cruaud ◽  
Jean-Yves Rasplus ◽  
Lillian Jennifer Rodriguez ◽  
Astrid Cruaud
Keyword(s):  
Quality Control ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Integrative Taxonomy ◽  
Miseq Sequencing ◽  
Dna Taxonomy ◽  
Cost Effective Method ◽  
Diversity Assessment ◽  
Pcr Conditions

ABSTRACTUntil now, the potential of NGS has been seldom realised for the construction of barcode reference libraries. Using a two-step PCR approach and MiSeq sequencing, we tested a cost-effective method and developed a custom workflow to simultaneously sequence multiple markers (COI, Cytb and EF, altogether 2kb) from hundreds of specimens. Interestingly, primers and PCR conditions used for Sanger sequencing did not require optimisation to construct MiSeq library. After completion of quality controls, 87% of the species and 76% of the specimens had valid sequences for the three markers. Nine specimens (3%) exhibited two divergent (up to 10%) sequence clusters. In 95% of the species, MiSeq and Sanger sequences obtained from the same samplings were similar. For the remaining 5%, species were paraphyletic or the sequences clustered into two divergent groups (>7%) on the final trees (Sanger + MiSeq). These problematic cases are difficult to explain but may represent coding NUMTS or heteroplasms. These results highlight the importance of performing quality control steps, working with expert taxonomists and using more than one marker for DNA-taxonomy or species diversity assessment. The power and simplicity of this method appears promising to build on existing experience, tools and resources while taking advantage of NGS.

scholarly journals Clinical Use of Next-Generation Sequencing in the Diagnosis of Wilson’s Disease

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Dániel Németh ◽  
Kristóf Árvai ◽  
Péter Horváth ◽  
János Pál Kósa ◽  
Bálint Tobiás ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Wilson’S Disease ◽  
Cost Effective ◽  
Wilson's Disease ◽  
Clinical Use ◽  
Next Generation ◽  
Time Saving ◽  
Cost Effective Method ◽  
Generation Sequencing

Objective. Wilson’s disease is a disorder of copper metabolism which is fatal without treatment. The great number of disease-causingATP7Bgene mutations and the variable clinical presentation of WD may cause a real diagnostic challenge. The emergence of next-generation sequencing provides a time-saving, cost-effective method for full sequencing of the wholeATP7Bgene compared to the traditional Sanger sequencing. This is the first report on the clinical use of NGS to examineATP7Bgene.Materials and Methods. We used Ion Torrent Personal Genome Machine in four heterozygous patients for the identification of the other mutations and also in two patients with no known mutation. One patient with acute on chronic liver failure was a candidate for acute liver transplantation. The results were validated by Sanger sequencing.Results. In each case, the diagnosis of Wilson’s disease was confirmed by identifying the mutations in both alleles within 48 hours. One novel mutation (p.Ala1270Ile) was found beyond the eight other known ones. The rapid detection of the mutations made possible the prompt diagnosis of WD in a patient with acute liver failure.Conclusions. According to our results we found next-generation sequencing a very useful, reliable, time-saving, and cost-effective method for diagnosing Wilson’s disease in selected cases.

scholarly journals Detection and Analysis of C-to-U RNA Editing in Rice Mitochondria-Encoded ORFs

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1277
Author(s):  
Peng Zheng ◽  
Dongxin Wang ◽  
Yuqing Huang ◽  
Hao Chen ◽  
Hao Du ◽  
...  
Keyword(s):  
Rna Editing ◽  
Sanger Sequencing ◽  
Open Reading Frames ◽  
Rna Modification ◽  
Rna Seq ◽  
Functional Consequence ◽  
Precise Information ◽  
Conserved Amino Acids ◽  
Reading Frames ◽  
Biological Context

Cytidine to uridine (C-to-U) RNA editing is an important type of substitutional RNA modification and is almost omnipresent in plant chloroplasts and mitochondria. In rice mitochondria, 491 C-to-U editing sites have been identified previously, and case studies have elucidated the function of several C-to-U editing sites in rice, but the functional consequence of most C-to-U alterations needs to be investigated further. Here, by means of Sanger sequencing and publicly available RNA-seq data, we identified a total of 569 C-to-U editing sites in rice mitochondria-encoded open reading frames (ORFs), 85.41% of these editing sites were observed on the first or the second base of a codon, resulting in the alteration of encoded amino acid. Moreover, we found some novel editing sites and several inaccurately annotated sites which may be functionally important, based on the highly conserved amino acids encoded by these edited codons. Finally, we annotated all 569 C-to-U RNA editing sites in their biological context. More precise information about C-to-U editing sites in rice mitochondria-encoded ORFs will facilitate our investigation on the function of C-to-U editing events in rice and also provide a valid benchmark from rice for the analysis of mitochondria C-to-U editing in other plant species.

scholarly journals Genome-Wide Identification of U-To-C RNA Editing Events for Nuclear Genes in Arabidopsis thaliana

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 635
Author(s):  
Ruchika ◽  
Chisato Okudaira ◽  
Matomo Sakari ◽  
Toshifumi Tsukahara
Keyword(s):  
Rna Editing ◽  
Sanger Sequencing ◽  
Developmental Stages ◽  
Nuclear Genes ◽  
Rna Seq ◽  
Pentatricopeptide Repeat ◽  
Stages Of Growth ◽  
Genome Wide ◽  
Ppr Genes ◽  
Plant Organelles

Cytosine-to-Uridine (C-to-U) RNA editing involves the deamination phenomenon, which is observed in animal nucleus and plant organelles; however, it has been considered the U-to-C is confined to the organelles of limited non-angiosperm plant species. Although previous RNA-seq-based analysis implied U-to-C RNA editing events in plant nuclear genes, it has not been broadly accepted due to inadequate confirmatory analyses. Here we examined the U-to-C RNA editing in Arabidopsis tissues at different developmental stages of growth. In this study, the high-throughput RNA sequencing (RNA-seq) of 12-day-old and 20-day-old Arabidopsis seedlings was performed, which enabled transcriptome-wide identification of RNA editing sites to analyze differentially expressed genes (DEGs) and nucleotide base conversions. The results showed that DEGs were expressed to higher levels in 12-day-old seedlings than in 20-day-old seedlings. Additionally, pentatricopeptide repeat (PPR) genes were also expressed at higher levels, as indicated by the log2FC values. RNA-seq analysis of 12-day- and 20-day-old Arabidopsis seedlings revealed candidates of U-to-C RNA editing events. Sanger sequencing of both DNA and cDNA for all candidate nucleotide conversions confirmed the seven U-to-C RNA editing sites. This work clearly demonstrated presence of U-to-C RNA editing for nuclear genes in Arabidopsis, which provides the basis to study the mechanism as well as the functions of the unique post-transcriptional modification.

scholarly journals CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals

2020 ◽  
Author(s):  
Efthymia Symeonidi ◽  
Julian Regalado ◽  
Rebecca Schwab ◽  
Detlef Weigel
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Repair Process ◽  
Wild Type ◽  
Isochorismate Synthase ◽  
Guide Rna ◽  
Cost Effective Method ◽  
Variable Efficiency

AbstractBackgroundGenome editing with the CRISPR/Cas9 system allows the user to mutate a targeted region of the genome using an endonuclease (Cas9) and an artificial single-guide RNA (sgRNA). Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to CRISPR/Cas9 mutagenesis. This process can be laborious, expensive and inefficient with conventional methods such as the T7E1 assay or Sanger sequencing. An alternative comprises methods for amplicon sequencing, but most available protocols do not include a facile way for high throughput generation of the samples for sequencing.ResultsIn this study we provide a full pipeline based on amplicon sequencing, CRISPR-finder. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. For example, we were able to analyze in one sequencing reaction over 900 Arabidopsis thaliana individuals whose genomes had been targeted with the CRISPR/Cas9 system.ConclusionsIn order to validate the potential of CRISPR-finder, we targeted the ISOCHORISMATE SYNTHASE 1 gene in A. thaliana using the CRISPR/Cas9 system. We successfully identified a mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and -efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

scholarly journals Multiplexed single-cell RNA-seq via transient barcoding for simultaneous expression profiling of various drug perturbations

2019 ◽  
Vol 5 (5) ◽  
pp. eaav2249 ◽  
Author(s):  
Dongju Shin ◽  
Wookjae Lee ◽  
Ji Hyun Lee ◽  
Duhee Bang
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cost Effective ◽  
Specific Gene ◽  
Rna Seq ◽  
Experimental Conditions ◽  
Cost Effective Method ◽  
Treatment Experiment ◽  
Single Cell Profiling ◽  
Multiple Samples

The development of high-throughput single-cell RNA sequencing (scRNA-seq) has enabled access to information about gene expression in individual cells and insights into new biological areas. Although the interest in scRNA-seq has rapidly grown in recent years, the existing methods are plagued by many challenges when performing scRNA-seq on multiple samples. To simultaneously analyze multiple samples with scRNA-seq, we developed a universal sample barcoding method through transient transfection with short barcode oligonucleotides. By conducting a species-mixing experiment, we have validated the accuracy of our method and confirmed the ability to identify multiplets and negatives. Samples from a 48-plex drug treatment experiment were pooled and analyzed by a single run of Drop-Seq. This revealed unique transcriptome responses for each drug and target-specific gene expression signatures at the single-cell level. Our cost-effective method is widely applicable for the single-cell profiling of multiple experimental conditions, enabling the widespread adoption of scRNA-seq for various applications.

scholarly journals A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

Plant Methods ◽  
2012 ◽  
Vol 8 (1) ◽  
pp. 41 ◽  
Author(s):  
Yun-Ru Chen ◽  
Yi Zheng ◽  
Bao Liu ◽  
Silin Zhong ◽  
Jim Giovannoni ◽  
...  
Keyword(s):  
Small Rna ◽  
Cost Effective ◽  
Library Preparation ◽  
Rna Seq ◽  
Rna Ligase ◽  
T4 Rna Ligase ◽  
Cost Effective Method

scholarly journals EditR: A novel base editing quantification software using Sanger sequencing

2017 ◽  
Author(s):  
Mitchell G. Kluesner ◽  
Derek A. Nedveck ◽  
Walker S. Lahr ◽  
John R. Garbe ◽  
Juan E. Abrahante ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Cytidine Deaminase ◽  
Cost Effective ◽  
Enzymatic Assays ◽  
Online Tool ◽  
Base Editing ◽  
Guide Rna ◽  
Desktop Application ◽  
Fusion Enzymes ◽  
Position Type

ABSTRACTCRISPR/Cas9-Cytidine deaminase fusion enzymes - termed Base Editors – allow targeted editing of genomic deoxcytidine to deoxthymidine (C→T) without the need for double stranded break induction. Base editors represent a paradigm-shift in gene editing technology, due to their unprecedented efficiency to mediate targeted, single-base conversion; however, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time consuming. To overcome these limitations, we developed a simple, cost effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed EditR. We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence (baseeditr.com). EditR is more accurate than enzymatic assays, and provides added insight to the position, type and efficiency of base editing. Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.

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