scholarly journals Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Luyi Tian ◽  
Jafar S. Jabbari ◽  
Rachel Thijssen ◽  
Quentin Gouil ◽  
Shanika L. Amarasinghe ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Single Cells ◽  
Transcript Level ◽  
Cell Types ◽  
Alternative Transcript ◽  
Long Read ◽  
Different Cell Types ◽  
Human And Mouse ◽  
Comprehensive Characterization

AbstractA modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.

scholarly journals Comprehensive characterization of single cell full-length isoforms in human and mouse with long-read sequencing

2020 ◽  
Author(s):  
Luyi Tian ◽  
Jafar S. Jabbari ◽  
Rachel Thijssen ◽  
Quentin Gouil ◽  
Shanika L. Amarasinghe ◽  
...  
Keyword(s):  
Data Integration ◽  
Single Cell ◽  
Ribosome Biogenesis ◽  
Single Cells ◽  
Transcript Level ◽  
Full Length ◽  
Alternative Transcript ◽  
Long Read ◽  
Comprehensive Characterization

AbstractAlternative splicing shapes the phenotype of cells in development and disease. Long-read RNA-sequencing recovers full-length transcripts but has limited throughput at the single-cell level. Here we developed single-cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to overcome these issues and perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, we performed the first comprehensive characterization of the full-length isoform landscape in single cells of different types and species and identified thousands of unannotated isoforms. We found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. Analysis at the transcript-level allowed data integration with scATAC-seq on individual promoters, improved correlation with protein expression data and linked mutations known to confer drug resistance to transcriptome heterogeneity. Our methods reveal previously unseen isoform complexity and provide a better framework for multi-omics data integration.

scholarly journals Single-cell RNA cap and tail sequencing (scRCAT-seq) reveals subtype-specific isoforms differing in transcript demarcation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Youjin Hu ◽  
Jiawei Zhong ◽  
Yuhua Xiao ◽  
Zheng Xing ◽  
Katherine Sheu ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Single Gene ◽  
Cell Types ◽  
Machine Learning Algorithms ◽  
Translation Efficiency ◽  
Transcription Start Sites ◽  
Long Read ◽  
Mrna Gene ◽  
Gene Isoforms

Abstract The differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Gene isoforms allow a single gene diverse functions across different cell types, and isoform dynamics allow different functions over time. However, methods to efficiently identify and quantify RNA isoforms genome-wide in single cells are still lacking. Here, we introduce single cell RNA Cap And Tail sequencing (scRCAT-seq), a method to demarcate the boundaries of isoforms based on short-read sequencing, with higher efficiency and lower cost than existing long-read sequencing methods. In conjunction with machine learning algorithms, scRCAT-seq demarcates RNA transcripts with unprecedented accuracy. We identified hundreds of previously uncharacterized transcripts and thousands of alternative transcripts for known genes, revealed cell-type specific isoforms for various cell types across different species, and generated a cell atlas of isoform dynamics during the development of retinal cones.

Biological Characteristics and Multilineage Differentiation of Kidney Mesenchymal Stem Cells Derived from the Tibetan Mastiff

2019 ◽  
Vol 9 (7) ◽  
pp. 904-913
Author(s):  
Bing Yan ◽  
Ruining Liang ◽  
Meng Ji ◽  
Qi-Qige Wuyun ◽  
Weijun Guan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Marker Gene ◽  
Cell Types ◽  
Immunofluorescence Staining ◽  
Marker Genes ◽  
Rt Pcr ◽  
Multipotent Stem Cells ◽  
Different Cell Types

Of all the significant researches that have taken place in isolation, culture and characterization of mesenchymal stem cells (MSCs), the field of kidney-derived mesenchymal stem cells (KMSCs) in Tibetan mastiff is still a blank. Therefore, the purpose of this study is to isolate, culture and characterize the Tibetan mastiff KMSCs. The KMSCs were successfully isolated from one-day year old Tibetan mastiff kidney, cultured for 16 passages and distinguished by two methods: immunofluorescence staining and RT-PCR. The Tibetan mastiff KMSCs expressed specific surface marker genes (VIM, CD44, FN1, CD90, CD109, CD73, FN1) and kidney marker gene PAX2. The proliferation ability of Tibetan mastiff KMSCs was measured through cell count and clonality. Furthermore, cells differentiated into different cell types (hepatocellular cells, osteogenic cells, adipogenic cells and chondrogenic cells) under special induced medium, and the marker genes of induced cells were identified with Immunofluorescence staining and RT-PCR. All of these results indicated that the Tibetan mastiff KMSCs were obtained successfully, which possessed certain characteristics of multipotent stem cells. Therefore, MSCs in Tibetan mastiff kidney hold potential for clinical applications for regenerative therapy and their further studies are waiting to be required to investigate their functions.

scholarly journals A splice variant acquiring an extra transcript leader region decreases the translation of glutamine synthetase gene

2003 ◽  
Vol 374 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Daesung SHIN ◽  
Sangjin PARK ◽  
Chankyu PARK
Keyword(s):  
Alternative Splicing ◽  
Glutamine Synthetase ◽  
Genomic Structure ◽  
Cell Types ◽  
Alternative Transcript ◽  
Exon Trapping ◽  
Exon 1 ◽  
Rapid Amplification ◽  
Altered Form ◽  
Human And Mouse

The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5′-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase–PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1′. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5′-untranslated region (UTR) containing the exon 1′. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin–Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5′-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5′-UTRs, demonstrating that the long form with extra 5′-UTR was translated 20- and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5′-UTRs were less efficient, showing 6–8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1′, the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5′-UTR is, to some extent, due to an abortive translation from the upstream ATG.

scholarly journals Towards a Comprehensive Variation Benchmark for Challenging Medically-Relevant Autosomal Genes

2021 ◽  
Author(s):  
Justin Wagner ◽  
Nathan D Olson ◽  
Lindsay Harris ◽  
Jennifer McDaniel ◽  
Haoyu Cheng ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Method Development ◽  
Clinical Applications ◽  
Accurate Analysis ◽  
Short Read ◽  
Short Read Sequencing ◽  
Repetitive Nature ◽  
Long Read ◽  
Comprehensive Characterization

The repetitive nature and complexity of multiple medically important genes make them intractable to accurate analysis, despite the maturity of short-read sequencing, resulting in a gap in clinical applications of genome sequencing. The Genome in a Bottle Consortium has provided benchmark variant sets, but these excluded some medically relevant genes due to their repetitiveness or polymorphic complexity. In this study, we characterize 273 of these 395 challenging autosomal genes that have multiple implications for medical sequencing. This extended, curated benchmark reports over 17,000 SNVs, 3,600 INDELs, and 200 SVs each for GRCh37 and GRCh38. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically important genes including CBS, CRYAA, and KCNE1. Our proposed solution improves variant recall in these genes from 8% to 100%. This benchmark will significantly improve the comprehensive characterization of these medically relevant genes and guide new method development.

scholarly journals Computational approaches towards reducing contamination in single-cell RNA-seq data

2020 ◽  
Author(s):  
Siamak Yousefi ◽  
Hao Chen ◽  
Jesse F. Ingels ◽  
Melinda S. McCarty ◽  
Arthur G. Centeno ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Real Life ◽  
Cell Types ◽  
Cell Capture ◽  
Rna Seq ◽  
Sequence Analyses ◽  
Cell Functions ◽  
Biological Interpretation ◽  
Different Cell Types

SUMMARYSingle cell RNA sequencing has enabled quantification of single cells and identification of different cell types and subtypes as well as cell functions in different tissues. Single cell RNA sequence analyses assume acquired RNAs correspond to cells, however, RNAs from contamination within the input data are also captured by these assays. The sequencing of background contamination as well as unwanted cells making their way to the final assay Potentially confound the correct biological interpretation of single cell transcriptomic data. Here we demonstrate two approaches to deal with background contamination as well as profiling of unwanted cells in the assays. We use three real-life datasets of whole-cell capture and nucleotide single-cell captures generated by Fluidigm and 10x technologies and show that these methods reduce the effect of contamination, strengthen clustering of cells and improves biological interpretation.

scholarly journals Mapping multicellular programs from single-cell profiles

2020 ◽  
Author(s):  
Livnat Jerby-Arnon ◽  
Aviv Regev
Keyword(s):  
Single Cell ◽  
Spatial Data ◽  
Spatial Information ◽  
Single Cells ◽  
Cell Types ◽  
Risk Genes ◽  
Health And Disease ◽  
Different Cell Types ◽  
Cellular Components ◽  
Acting In Concert

ABSTRACTTissue homeostasis relies on orchestrated multicellular circuits, where interactions between different cell types dynamically balance tissue function. While single-cell genomics identifies tissues’ cellular components, deciphering their coordinated action remains a major challenge. Here, we tackle this problem through a new framework of multicellular programs: combinations of distinct cellular programs in different cell types that are coordinated together in the tissue, thus forming a higher order functional unit at the tissue, rather than only cell, level. We develop the open-access DIALOGUE algorithm to systematically uncover such multi-cellular programs not only from spatial data, but even from tissue dissociated and profiled as single cells, e.g., by single-cell RNA-Seq. Tested on spatial transcriptomes from the mouse hypothalamus, DIALOGUE recovered spatial information, predicted the properties of a cell’s environment only based on its transcriptome, and identified multicellular programs that mark animal behavior. Applied to brain samples and colon biopsies profiled by scRNA-Seq, DIALOGUE identified multicellular configurations that mark Alzheimer’s disease and ulcerative colitis (UC), including a program spanning five cell types that is predictive of response to anti-TNF therapy in UC patients and enriched for UC risk genes from GWAS, each acting in different cell types, but all cells acting in concert. Taken together, our study provides a novel conceptual and methodological framework to unravel multicellular regulation in health and disease.

scholarly journals Unfolding and identification of membrane proteins from native cell membranes

2019 ◽  
Author(s):  
Nicola Galvanetto ◽  
Sourav Maity ◽  
Nina Ilieva ◽  
Zhongjie Ye ◽  
Alessandro Laio ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Single Cells ◽  
Total Content ◽  
Cell Types ◽  
Single Cell Level ◽  
Cell Level ◽  
Single Molecule Force Spectroscopy ◽  
Native Cell ◽  
Different Cell Types

AbstractIs the mechanical unfolding of proteins just a technological feat applicable only to synthetic preparations or can it provide useful information even for real biological samples? Here, we describe a pipeline for analyzing native membranes based on high throughput single-molecule force spectroscopy. The protocol includes a technique for the isolation of the plasma membrane of single cells. Afterwards, one harvests hundreds of thousands SMSF traces from the sample. Finally, one characterizes and identifies the embedded membrane proteins. This latter step is the cornerstone of our approach and involves combining, within a Bayesian framework, the information of the shape of the SMFS Force-distance which are observed more frequently, with the information from Mass Spectrometry and from proteomic databases (Uniprot, PDB). We applied this method to four cell types where we classified the unfolding of 5-10% of their total content of membrane proteins. The ability to mechanically probe membrane proteins directly in their native membrane enables the phenotyping of different cell types with almost single-cell level of resolution.

scholarly journals A screen for mutants deficient in coronatine-mediated suppression of root immunity identifies Arabidopsis SDA1 as a novel integrator of immunity and phytohormone signaling

2021 ◽  
Author(s):  
Yi Song ◽  
Xue-Cheng Zhang ◽  
Yichun Qiu ◽  
Annika Briggs ◽  
Yves Millet ◽  
...  
Keyword(s):  
Fungal Pathogen ◽  
Ribosome Biogenesis ◽  
Negative Regulator ◽  
Root Growth Inhibition ◽  
Genetic Screens ◽  
Negative Regulators ◽  
Enhanced Resistance ◽  
Foliar Pathogen ◽  
Comprehensive Characterization

Despite the importance of the root immune system in the interaction with rhizosphere microbes, the majority of genetic screens for immunity regulators have been performed in leaves. A previous screen identified 27 hsm (hormone-mediated suppression of MAMP-triggered immunity) mutants that are impaired in jasmonic acid (JA)-mediated suppression of pattern-triggered immunity (PTI) in roots. Here we characterized 16 of the hsm mutants that retain JA sensitivity and are potential negative regulators of root immunity. We found that the majority of hsm mutants show enhanced resistance to Fusarium, a root fungal pathogen; however, only a subset are more resistant to a foliar pathogen. Surprisingly, 12 of 16 hsm mutants are also impaired in abscisic acid (ABA)-mediated suppression of PTI, suggesting a largely shared pathway between JA- and ABA-mediated immune suppression in roots. Although all hsm mutants are insensitive to JA-mediated suppression of root immunity, hsm4 shows hypersensitivity to JA-mediated root growth inhibition and JA-induced gene expression. Consistently, hsm4 is more resistant to leaf pathogens, suggesting that HSM4 is a negative regulator of both root and leaf immunity. Hsm4 was mapped to a mutation in a conserved ARM-repeat protein homologous to yeast SDA1, which has been reported to regulate 60S ribosome biogenesis. As translational reprogramming is a critical layer of immune regulation, this work suggests that AtSDA1 is a novel negative translational regulator of immunity. Additionally, a comprehensive characterization of all 16 hsm mutants provides a genetic toolkit to identify novel mechanisms that regulate root immunity.

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