scholarly journals Obstacles to Studying Alternative Splicing Using scRNA-seq

2019 ◽  
Author(s):  
Jennifer Westoby ◽  
Pavel Artemov ◽  
Martin Hemberg ◽  
Anne Ferguson-Smith
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
High Rate ◽  
Model Organisms ◽  
Rna Seq ◽  
Simulation Based ◽  
Simulation Results ◽  
The Impact ◽  
Isoform Quantification ◽  
Low Rate

AbstractBackgroundEarly single-cell RNA-seq (scRNA-seq) studies suggested that it was unusual to see more than one isoform being produced from a gene in a single cell, even when multiple isoforms were detected in matched bulk RNA-seq samples. However, these studies generally did not consider the impact of dropouts or isoform quantification errors, potentially confounding the results of these analyses.ResultsIn this study, we take a simulation based approach in which we explicitly account for dropouts and isoform quantification errors. We use our simulations to ask to what extent it is possible to study alternative splicing using scRNA-seq. Additionally, we ask what limitations must be overcome to make splicing analysis feasible. We find that the high rate of dropouts associated with scRNA-seq is a major obstacle to studying alternative splicing. In mice and other well established model organisms, the relatively low rate of isoform quantification errors poses a lesser obstacle to splicing analysis. We find that different models of isoform choice meaningfully change our simulation results.ConclusionsTo accurately study alternative splicing with single-cell RNA-seq, a better understanding of isoform choice and the errors associated with scRNA-seq is required. An increase in the capture efficiency of scRNA-seq would also be beneficial. Until some or all of the above are achieved, we do not recommend attempting to resolve isoforms in individual cells using scRNA-seq.

scholarly journals Simulation-based benchmarking of isoform quantification in single-cell RNA-seq

2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Jennifer Westoby ◽  
Marcela Sjöberg Herrera ◽  
Anne C. Ferguson-Smith ◽  
Martin Hemberg
Keyword(s):  
Single Cell ◽  
Rna Seq ◽  
Simulation Based ◽  
Isoform Quantification

scholarly journals Simulation based benchmarking of isoform quantification in single-cell RNA-seq

2018 ◽  
Author(s):  
Jennifer Westoby ◽  
Marcela Sjoberg ◽  
Anne Ferguson-Smith ◽  
Martin Hemberg
Keyword(s):  
Single Cell ◽  
Confounding Factor ◽  
Simulated Data ◽  
Real Data ◽  
Mixed Population ◽  
Rna Seq ◽  
Simulation Based ◽  
Biological Insight ◽  
Isoform Quantification

AbstractSingle-cell RNA-seq has the potential to facilitate isoform quantification as the confounding factor of a mixed population of cells is eliminated. We carried out a benchmark for five popular isoform quantification tools. Performance was generally good when run on simulated data based on SMARTer and SMART-seq2 data, but was poor for simulated Drop-seq data. Importantly, the reduction in performance for single-cell RNA-seq compared with bulk RNA-seq was small. An important biological insight comes from our analysis of real data which showed that genes that express two isoforms in bulk RNA-seq predominantly express one or neither isoform in individual cells.

scholarly journals Single cell analysis reveals the impact of age and maturation stage on the human oocyte transcriptome

2020 ◽  
Author(s):  
Silvia Llonch ◽  
Montserrat Barragán ◽  
Paula Nieto ◽  
Anna Mallol ◽  
Marc Elosua-Bayes ◽  
...  
Keyword(s):  
Single Cell ◽  
Chromosome Segregation ◽  
Maternal Age ◽  
Maturation Stage ◽  
P Value ◽  
Rna Seq ◽  
Transcript Levels ◽  
Human Oocytes ◽  
The Impact

AbstractStudy questionTo which degree does maternal age affect the transcriptome of human oocytes at the germinal vesicle (GV) stage or at metaphase II after maturation in vitro (IVM-MII)?Summary answerWhile the oocytes’ transcriptome is predominantly determined by maturation stage, transcript levels of genes related to chromosome segregation, mitochondria and RNA processing are affected by age after in vitro maturation of denuded oocytes.What is known alreadyFemale fertility is inversely correlated with maternal age due to both a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, and the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown GV and IVM-MII oocytes from women of varying reproductive age.Study design, size, durationIn this prospective molecular study, 37 women were recruited from May 2018 to June 2019. The mean age was 28.8 years (SD=7.7, range 18-43). A total of 72 oocytes were included in the study at GV stage after ovarian stimulation, and analyzed as GV (n=40) and in vitro matured oocytes (IVM-MII; n=32).Participants/materials, setting, methodsDenuded oocytes were included either as GV at the time of ovum pick-up or as IVM-MII after in vitro maturation for 30 hours in G2™ medium, and processed for transcriptomic analysis by single-cell RNA-seq using the Smart-seq2 technology. Cluster and maturation stage marker analysis were performed using the Seurat R package. Genes with an average fold change greater than 2 and a p-value < 0.01 were considered maturation stage markers. A Pearson correlation test was used to identify genes whose expression levels changed progressively with age. Those genes presenting a correlation value (R) >= |0.3| and a p-value < 0.05 were considered significant.Main results and the role of chanceFirst, by exploration of the RNA-seq data using tSNE dimensionality reduction, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4,445 and 324 putative marker genes, respectively. Next we identified genes, for which RNA levels either progressively increased or decreased with age. This analysis was performed independently for GV and IVM-MII oocytes. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1,219 genes) than in GV oocytes (596 genes). In particular, we found that genes involved in chromosome segregation and RNA splicing significantly increase in transcript levels with age, while genes related to mitochondrial activity present lower transcript levels with age. Gene regulatory network analysis revealed potential upstream master regulator functions for genes whose transcript levels present positive (GPBP1, RLF, SON, TTF1) or negative (BNC1, THRB) correlation with age.Limitations, reasons for cautionIVM-MII oocytes used in this study were obtained after in vitro maturation of denuded GV oocytes, therefore, their transcriptome might not be fully representative of in vivo matured MII oocytes.The Smart-seq2 methodology used in this study detects polyadenylated transcripts only and we could therefore not assess non-polyadenylated transcripts.Wider implications of the findingsOur analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript levels with age, particularly in IVM-MII oocytes. Especially affected by age were genes related to chromosome segregation and mitochondrial function, pathways known to be involved in oocyte ageing. Our study thereby suggests that misregulation of chromosome segregation and mitochondrial pathways also at the RNA-level might contribute to the age-related quality decline in human oocytes.Study funding/competing interest(s)This study was funded by the AXA research fund, the European commission, intramural funding of Clinica EUGIN, the Spanish Ministry of Science, Innovation and Universities, the Catalan Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) and by contributions of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and to the “Centro de Excelencia Severo Ochoa”.The authors have no conflict of interest to declare.

Analysis on Coupling Faults Induced Vibration of Hydropower Unit

2013 ◽  
Vol 397-400 ◽  
pp. 397-400
Author(s):  
Xing Liang ◽  
De Hui Liu ◽  
Jia Zhang Gui ◽  
Yun Min Xie ◽  
Jin Guang Huang
Keyword(s):  
Orthogonal Test ◽  
High Rate ◽  
Impact Factors ◽  
Rotor Vibration ◽  
Coupling Faults ◽  
Parallel Misalignment ◽  
Mass Eccentricity ◽  
Simulation Results ◽  
The Impact ◽  
Misalignment Fault

A mathematical model of hydropower units shaft is established to characterize the rotor motion with coupling faults which compose of mass eccentricity fault, parallel misalignment fault and angle misalignment fault, and then the simulation results is employed to analyze the impact weight of faults above for vibration and rubbing based on orthogonal test. The research showed that: the major-minor order of impact factors are mass eccentricity, parallel misalignment and angle misalignment. Additionally, the rotating rate is also related to the rotor vibration, and the high rate is easy to induce the rubbing under coupling faults.

scholarly journals Simulation and Prediction of the Groundwater Pollution Caused by the Leakage from a Sewage Plant in an Aluminum Factory Under the Action of Ocean Tide Support

2021 ◽  
Author(s):  
Yu Zhang ◽  
Junkang Lan
Keyword(s):  
Groundwater Pollution ◽  
Permeability Coefficient ◽  
Ocean Tide ◽  
Pollution Transport ◽  
Groundwater Environment ◽  
Groundwater Aquifer ◽  
Simulation Based ◽  
Simulation Results ◽  
Groundwater Flow Field ◽  
The Impact

In order to predict the impact of wastewater from an aluminum plant treatment station on the groundwater environment under abnormal conditions (i.e., sewage leakage accident). Through the investigation of hydrogeological conditions, and then the permeability coefficient of the aquifer was measured through borehole injection tests. Finally, the groundwater pollution transport halo was obtained by numerical simulation based GMS software. The simulation results showed that the groundwater aquifer will be seriously polluted by COD and fluoride (F-) after the sudden sewage seepage accident. What’s more, the simulation results showed that the pollution concentration is getting higher and higher with time, which is analyzed to be caused by the small permeability of the water-bearing medium in the aquifer and the groundwater flow field was supported by seawater tide.

scholarly journals Splatter: simulation of single-cell RNA sequencing data

2017 ◽  
Author(s):  
Luke Zappia ◽  
Belinda Phipson ◽  
Alicia Oshlack
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Real Data ◽  
Cell Types ◽  
Rna Seq ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
Simulation Based ◽  
Single Cell Rna Sequencing ◽  
Multiple Cell

AbstractAs single-cell RNA sequencing technologies have rapidly developed, so have analysis methods. Many methods have been tested, developed and validated using simulated datasets. Unfortunately, current simulations are often poorly documented, their similarity to real data is not demonstrated, or reproducible code is not available.Here we present the Splatter Bioconductor package for simple, reproducible and well-documented simulation of single-cell RNA-seq data. Splatter provides an interface to multiple simulation methods including Splat, our own simulation, based on a gamma-Poisson distribution. Splat can simulate single populations of cells, populations with multiple cell types or differentiation paths.

scholarly journals Per-sample standardization and asymmetric winsorization lead to accurate clustering of RNA-seq expression profiles

2021 ◽  
Author(s):  
Davide Risso ◽  
Stefano Maria Pagnotta
Keyword(s):  
Single Cell ◽  
Expression Profiles ◽  
Unsupervised Clustering ◽  
Supplementary Information ◽  
Supplementary Data ◽  
Rna Seq ◽  
Data Transformations ◽  
The Impact

Abstract Motivation Data transformations are an important step in the analysis of RNA-seq data. Nonetheless, the impact of transformation on the outcome of unsupervised clustering procedures is still unclear. Results Here, we present an Asymmetric Winsorization per Sample Transformation (AWST), which is robust to data perturbations and removes the need for selecting the most informative genes prior to sample clustering. Our procedure leads to robust and biologically meaningful clusters both in bulk and in single-cell applications. Availability The AWST method is available at https://github.com/drisso/awst. The code to reproduce the analyses is available at https://github.com/drisso/awst\_analysis. Supplementary information Supplementary data are available at Bioinformatics online.

Glutton: large-scale integration of non-model organism transcriptome data for comparative analysis

2016 ◽  
Author(s):  
Alan Medlar ◽  
Laura Laakso ◽  
Andreia Miraldo ◽  
Ari Löytynoja
Keyword(s):  
Comparative Analysis ◽  
Large Scale ◽  
De Novo ◽  
Sequence Data ◽  
Model Organism ◽  
Model Organisms ◽  
Rna Seq ◽  
Reference Species ◽  
Wide Range ◽  
The Impact

AbstractHigh-throughput RNA-seq data has become ubiquitous in the study of non-model organisms, but its use in comparative analysis remains a challenge. Without a reference genome for mapping, sequence data has to be de novo assembled, producing large numbers of short, highly redundant contigs. Preparing these assemblies for comparative analyses requires the removal of redundant isoforms, assignment of orthologs and converting fragmented transcripts into gene alignments. In this article we present Glutton, a novel tool to process transcriptome assemblies for downstream evolutionary analyses. Glutton takes as input a set of fragmented, possibly erroneous transcriptome assemblies. Utilising phylogeny-aware alignment and reference data from a closely related species, it reconstructs one transcript per gene, finds orthologous sequences and produces accurate multiple alignments of coding sequences. We present a comprehensive analysis of Glutton’s performance across a wide range of divergence times between study and reference species. We demonstrate the impact choice of assembler has on both the number of alignments and the correctness of ortholog assignment and show substantial improvements over heuristic methods, without sacrificing correctness. Finally, using inference of Darwinian selection as an example of downstream analysis, we show that Glutton-processed RNA-seq data give results comparable to those obtained from full length gene sequences even with distantly related reference species. Glutton is available from http://wasabiapp.org/software/glutton/ and is licensed under the GPLv3.

scholarly journals A double-reprocessing high-level disinfection protocol does not eliminate positive cultures from the elevators of duodenoscopes

Endoscopy ◽  
2017 ◽  
Vol 50 (06) ◽  
pp. 588-596 ◽  
Author(s):  
Douglas Rex ◽  
Marnie Sieber ◽  
Glen Lehman ◽  
Douglas Webb ◽  
Bryan Schmitt ◽  
...  
Keyword(s):  
Positive Culture ◽  
High Rate ◽  
Pathogenic Potential ◽  
Entire Process ◽  
Third Phase ◽  
Serious Infection ◽  
Study Interval ◽  
High Level ◽  
The Impact ◽  
Low Rate

Abstract Background and study aim Duodenoscopes have been the source of serious infection, despite correct performance of high-level disinfection (HLD). This study aimed to observe the impact of performing HLD twice on the rate of positive cultures from duodenoscope elevators. Methods We performed double HLD (DHLD; i. e. complete manual cleaning followed by automated reprocessing, with the entire process repeated) and then randomly cultured the elevators of our duodenoscopes on about 30 % of occasions. Results DHLD was associated with positive elevator cultures for any microorganism in 9.4 % of cases, with a 0.8 % rate of known pathogens (627 cultures) between May 2015 and February 2016. After February 2016, and in association with changing the precleaning fluid, as well as use of a new FDA-recommended cleaning brush, the rate of positive cultures for any microorganism after DHLD was 4.8 % and 0.2 % for known pathogens (420 cultures). In a third phase, characterized by a change in personnel performing DHLD and retirement of a duodenoscope with a high rate of positive cultures, the rate of positive cultures for any microorganism was 4.9 % (783 cultures) and the rate of positive culture for known pathogens was 0.3 %. To our knowledge, no duodenoscope transmission of infection occurred during the study interval. Conclusions DHLD resulted in a low rate of positive cultures for known pathogens and for organisms of low pathogenic potential, but did not eliminate these, from duodenoscope elevators. Additional improvements in HLD protocols and/or duodenoscope design are needed.

scholarly journals LIQA: long-read isoform quantification and analysis

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yu Hu ◽  
Li Fang ◽  
Xuelian Chen ◽  
Jiang F. Zhong ◽  
Mingyao Li ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Myeloid Leukemia ◽  
Expectation Maximization Algorithm ◽  
Read Length ◽  
Specific Gene ◽  
Rna Seq ◽  
Sequencing Data ◽  
Long Read ◽  
Isoform Quantification ◽  
Universal Human Reference

AbstractLong-read RNA sequencing (RNA-seq) technologies can sequence full-length transcripts, facilitating the exploration of isoform-specific gene expression over short-read RNA-seq. We present LIQA to quantify isoform expression and detect differential alternative splicing (DAS) events using long-read direct mRNA sequencing or cDNA sequencing data. LIQA incorporates base pair quality score and isoform-specific read length information in a survival model to assign different weights across reads, and uses an expectation-maximization algorithm for parameter estimation. We apply LIQA to long-read RNA-seq data from the Universal Human Reference, acute myeloid leukemia, and esophageal squamous epithelial cells and demonstrate its high accuracy in profiling alternative splicing events.

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