scholarly journals Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq

2015 ◽  
Vol 15 (1) ◽  
Author(s):  
Sheena L Faherty ◽  
C Ryan Campbell ◽  
Peter A Larsen ◽  
Anne D Yoder
Keyword(s):  
Gene Profiling ◽  
Rna Seq ◽  
Whole Transcriptome Amplification ◽  
Whole Transcriptome

scholarly journals Impact of RNA Extraction and Target Capture Methods on RNA Sequencing Using Formalin-Fixed, Paraffin Embedded Tissues

2019 ◽  
Author(s):  
Christopher A. Hilker ◽  
Aditya V. Bhagwate ◽  
Jin Sung Jang ◽  
Jeffrey G Meyer ◽  
Asha A. Nair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Seq ◽  
Gene Detection ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

AbstractFormalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

scholarly journals Plasmodium parasites mount an arrest response to dihydroartemisinin, as revealed by whole transcriptome shotgun sequencing (RNA-seq) and microarray study

BMC Genomics ◽  
2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Philip J. Shaw ◽  
Sastra Chaotheing ◽  
Pavita Kaewprommal ◽  
Jittima Piriyapongsa ◽  
Chayaphat Wongsombat ◽  
...  
Keyword(s):  
Shotgun Sequencing ◽  
Rna Seq ◽  
Microarray Study ◽  
Whole Transcriptome

scholarly journals Stranded Whole Transcriptome RNA‐Seq for All RNA Types

2015 ◽  
Vol 84 (1) ◽  
Author(s):  
David F.B. Miller ◽  
Pearlly X. Yan ◽  
Fang Fang ◽  
Aaron Buechlein ◽  
James B. Ford ◽  
...  
Keyword(s):  
Rna Seq ◽  
Whole Transcriptome

scholarly journals Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification

PLoS ONE ◽  
2019 ◽  
Vol 14 (8) ◽  
pp. e0216442 ◽  
Author(s):  
Franziska C. Durst ◽  
Ana Grujovic ◽  
Iris Ganser ◽  
Martin Hoffmann ◽  
Peter Ugocsai ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Transcript Quantification ◽  
Whole Transcriptome Amplification ◽  
Whole Transcriptome

scholarly journals Screening of Virulence-Related Transcriptional Regulators in Streptococcus suis

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 972
Author(s):  
Liang Liu ◽  
Qiang Zhang ◽  
Zhongmin Xu ◽  
Bo Chen ◽  
Anding Zhang ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Streptococcus Suis ◽  
Transcriptional Regulators ◽  
Rna Seq ◽  
Related Factors ◽  
Transcriptional Regulatory ◽  
Mutant Strains ◽  
Whole Transcriptome

Streptococcus suis (S.suis) is an important zoonotic pathogen that causes many severe diseases in pigs and humans. Virulence-related transcriptional regulators have been widely reported in pathogenic microorganisms, but only a few have been identified in S.suis. Our aim was to screen virulence-related transcriptional regulators in S.suis. A total of 89 such genes were predicted in the S.suis genome, of which 22 were up-regulated and 18 were down-regulated during S.suis infection in mice. To evaluate the roles of these differentially expressed factors in S.suis virulence, deletion mutants were constructed, and 10 mutants were successfully obtained. Among these genes, the deletion of comR, sitR, or sxvR caused significantly decreased virulence in mice, compared to that with the wild-type strain. Moreover, the survival of ΔcomR, ΔsitR, and ΔsxvR mutant strains in blood was significantly reduced both in vitro and in vivo. Furthermore, their pro-inflammatory abilities were also obviously decreased in vivo. The regulatory mechanisms of comR, sitR, and sxvR were then analyzed by whole transcriptome RNA sequencing (RNA-Seq). Results indicated that the absence of comR induced the down-regulation of 17 virulence factors or virulence-related factors, including genes involved in the synthesis of capsules, oxidative stress tolerance, immune evasion, and cell division. Furthermore, three and two virulence factors or virulence-related factors were down-regulated upon deletion of sitR and sxvR, respectively. Thus, this study reports the discovery of three virulence-associated transcriptional regulatory factors in S.suis. These factors could ultimately be targeted to control infection caused by these bacteria.

scholarly journals CoCo: RNA-seq read assignment correction for nested genes and multimapped reads

2019 ◽  
Vol 35 (23) ◽  
pp. 5039-5047 ◽  
Author(s):  
Gabrielle Deschamps-Francoeur ◽  
Vincent Boivin ◽  
Sherif Abou Elela ◽  
Michelle S Scott
Keyword(s):  
Supplementary Information ◽  
Rna Seq ◽  
Non Coding Rna ◽  
Abundance Estimates ◽  
Gene Coverage ◽  
Nested Genes ◽  
Quantification Accuracy ◽  
Whole Transcriptome Analysis ◽  
Whole Transcriptome ◽  
Generation Sequencing

Abstract Motivation Next-generation sequencing techniques revolutionized the study of RNA expression by permitting whole transcriptome analysis. However, sequencing reads generated from nested and multi-copy genes are often either misassigned or discarded, which greatly reduces both quantification accuracy and gene coverage. Results Here we present count corrector (CoCo), a read assignment pipeline that takes into account the multitude of overlapping and repetitive genes in the transcriptome of higher eukaryotes. CoCo uses a modified annotation file that highlights nested genes and proportionally distributes multimapped reads between repeated sequences. CoCo salvages over 15% of discarded aligned RNA-seq reads and significantly changes the abundance estimates for both coding and non-coding RNA as validated by PCR and bedgraph comparisons. Availability and implementation The CoCo software is an open source package written in Python and available from http://gitlabscottgroup.med.usherbrooke.ca/scott-group/coco. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals De novo transcriptomes of 14 gammarid individuals for proteogenomic analysis of seven taxonomic groups

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Yannick Cogne ◽  
Davide Degli-Esposti ◽  
Olivier Pible ◽  
Duarte Gouveia ◽  
Adeline François ◽  
...  
Keyword(s):  
Aquatic Ecosystems ◽  
Molecular Diversity ◽  
De Novo ◽  
Model Organisms ◽  
Rna Seq ◽  
Specific Expression ◽  
Gammarus Fossarum ◽  
Male Individual ◽  
Taxonomic Groups ◽  
Whole Transcriptome

Abstract Gammarids are amphipods found worldwide distributed in fresh and marine waters. They play an important role in aquatic ecosystems and are well established sentinel species in ecotoxicology. In this study, we sequenced the transcriptomes of a male individual and a female individual for seven different taxonomic groups belonging to the two genera Gammarus and Echinogammarus: Gammarus fossarum A, G. fossarum B, G. fossarum C, Gammarus wautieri, Gammarus pulex, Echinogammarus berilloni, and Echinogammarus marinus. These taxa were chosen to explore the molecular diversity of transcribed genes of genotyped individuals from these groups. Transcriptomes were de novo assembled and annotated. High-quality assembly was confirmed by BUSCO comparison against the Arthropod dataset. The 14 RNA-Seq-derived protein sequence databases proposed here will be a significant resource for proteogenomics studies of these ecotoxicologically relevant non-model organisms. These transcriptomes represent reliable reference sequences for whole-transcriptome and proteome studies on other gammarids, for primer design to clone specific genes or monitor their specific expression, and for analyses of molecular differences between gammarid species.

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