scholarly journals Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

2019 ◽  
Author(s):  
Celine Everaert ◽  
Hetty Helsmoortel ◽  
Anneleen Decock ◽  
Eva Hulstaert ◽  
Ruben Van Paemel ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Extracellular Vesicles ◽  
Platelet Rich Plasma ◽  
Rna Seq ◽  
Total Rna ◽  
Rna Molecules ◽  
Rna Profiling ◽  
Wide Range ◽  
Read Distribution ◽  
Free Plasma

AbstractRNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

scholarly journals Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Celine Everaert ◽  
Hetty Helsmoortel ◽  
Anneleen Decock ◽  
Eva Hulstaert ◽  
Ruben Van Paemel ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Extracellular Vesicles ◽  
Platelet Rich Plasma ◽  
Rna Seq ◽  
Total Rna ◽  
Rna Molecules ◽  
Rna Profiling ◽  
Wide Range ◽  
Read Distribution ◽  
Free Plasma

AbstractRNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals Emerging Roles of Estrogen-Regulated Enhancer and Long Non-Coding RNAs

2020 ◽  
Vol 21 (10) ◽  
pp. 3711
Author(s):  
Melina J. Sedano ◽  
Alana L. Harrison ◽  
Mina Zilaie ◽  
Chandrima Das ◽  
Ramesh Choudhari ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Expression Patterns ◽  
Biological Significance ◽  
Rna Seq ◽  
Biological Functions ◽  
Protein Coding ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Genome Wide ◽  
Non Coding Rnas

Genome-wide RNA sequencing has shown that only a small fraction of the human genome is transcribed into protein-coding mRNAs. While once thought to be “junk” DNA, recent findings indicate that the rest of the genome encodes many types of non-coding RNA molecules with a myriad of functions still being determined. Among the non-coding RNAs, long non-coding RNAs (lncRNA) and enhancer RNAs (eRNA) are found to be most copious. While their exact biological functions and mechanisms of action are currently unknown, technologies such as next-generation RNA sequencing (RNA-seq) and global nuclear run-on sequencing (GRO-seq) have begun deciphering their expression patterns and biological significance. In addition to their identification, it has been shown that the expression of long non-coding RNAs and enhancer RNAs can vary due to spatial, temporal, developmental, or hormonal variations. In this review, we explore newly reported information on estrogen-regulated eRNAs and lncRNAs and their associated biological functions to help outline their markedly prominent roles in estrogen-dependent signaling.

scholarly journals Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles

2020 ◽  
Author(s):  
A Koch ◽  
T Schlemmer ◽  
L Höfle ◽  
BT Werner ◽  
C Preußer ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Host Cells ◽  
Target Sequence ◽  
Rna Seq ◽  
Leaf Extracts ◽  
Transgenic Expression ◽  
Rna Molecules ◽  
Expression Host

AbstractSmall (s)RNA molecules are crucial factors in the communication between hosts and their interacting pathogens, where they function as effectors that can modulate both host defense and microbial virulence/pathogenicity through a mechanism termed cross-kingdom RNA interference (ckRNAi). Consistent with this recent knowledge, sRNAs and their double-stranded (ds)RNA precursors have been adopted to control diseases in crop plants through transgenic expression (host-induced gene silencing, HIGS) or exogenous application (spray-induced gene silencing, SIGS). While these strategies proved to be effective, the mechanism of RNA transfer at the plant - pathogen interface is widely unresolved. Here we show that extracellular vesicles (EVs) purified from Arabidopsis (Arabidopsis thaliana) leaf extracts and apoplastic fluids contain transgene-derived sRNAs. EVs from plants expressing CYP3RNA, a 791 nt long dsRNA, which was originally designed to target the three CYP51 genes of the fungal pathogen Fusarium graminearum, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. Notably, the EVs cargo retained the same CYP3RNA-derived siRNA profile as the respective leaf extracts, suggesting that there was no selective uptake of specific artificial sRNAs into EVs. In addition, mutants of the ESCRT-III complex were impaired in HIGS further indicating that endosomal vesicle trafficking supports transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells. Further supporting the relevance of EV-mediated transport of sRNA, we demonstrate that HIGS plants, expressing a 100 nt dsRNA-target-sequence identified via EV-sRNA-seq of CYP3RNA Arabidopsis, confers strong resistance to F. graminearum. Together, these findings support the view that EVs are key mediators in the transport of HIGS-related sRNAs to reduce the virulence of interacting fungal pathogens during host-pathogen interaction.

scholarly journals SPsimSeq: semi-parametric simulation of bulk and single cell RNA sequencing data

2019 ◽  
Author(s):  
Alemu Takele Assefa ◽  
Jo Vandesompele ◽  
Olivier Thas
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Empirical Distribution ◽  
Supplementary Information ◽  
Rna Seq ◽  
Sequencing Data ◽  
Actual Distribution ◽  
Wide Range ◽  
Single Cell Rna Sequencing

SummarySPsimSeq is a semi-parametric simulation method for bulk and single cell RNA sequencing data. It simulates data from a good estimate of the actual distribution of a given real RNA-seq dataset. In contrast to existing approaches that assume a particular data distribution, our method constructs an empirical distribution of gene expression data from a given source RNA-seq experiment to faithfully capture the data characteristics of real data. Importantly, our method can be used to simulate a wide range of scenarios, such as single or multiple biological groups, systematic variations (e.g. confounding batch effects), and different sample sizes. It can also be used to simulate different gene expression units resulting from different library preparation protocols, such as read counts or UMI counts.Availability and implementationThe R package and associated documentation is available from https://github.com/CenterForStatistics-UGent/SPsimSeq.Supplementary informationSupplementary data are available at bioRχiv online.

scholarly journals RNA sequencing by direct tagmentation of RNA/DNA hybrids

2020 ◽  
Vol 117 (6) ◽  
pp. 2886-2893 ◽  
Author(s):  
Lin Di ◽  
Yusi Fu ◽  
Yue Sun ◽  
Jie Li ◽  
Lu Liu ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Single Cells ◽  
Transcriptome Profiling ◽  
Initial Material ◽  
Library Preparation ◽  
Rna Seq ◽  
Cdna Synthesis ◽  
Complementary Dna ◽  
Dna Analyses ◽  
Wide Range

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

scholarly journals Comparing total RNA sequencing and metagenomics pipelines for multi-domain taxonomic profiling: implications for ecological assessments

2021 ◽  
Vol 4 ◽  
Author(s):  
Christopher Hempel ◽  
Julia Harvie ◽  
Jose Hleap Lozano ◽  
Natalie Wright ◽  
Sarah Adamowicz ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Rna Sequencing ◽  
Sample Type ◽  
Rna Seq ◽  
Total Rna ◽  
Taxonomic Profiling ◽  
Dna And Rna ◽  
Accuracy And Precision ◽  
The Status ◽  
Unicellular Organisms

Ecological assessments are necessary to evaluate the status of our deteriorating ecosystems, however, assessment methods traditionally omit most microbes because unicellular organisms are challenging to identify. This omission is not ideal, as microbes might be better indicators for changes in environmental conditions than taxa traditionally used. DNA- and RNA-based techniques are increasingly applied for ecological assessments to overcome this challenge but require more testing and optimization. In this study, we compare metagenomics and total RNA sequencing (total RNA-Seq) for their taxonomic profiling performance for microbial communities. We applied both techniques on two sample sets, 1) a commercially available microbial mock community consisting of eight bacterial and two eukaryotic species, and 2) a display tank water sample. We processed the data using 1,532 bioinformatics pipelines and evaluated each workflow, i.e., the combination of sample type (metagenomics or total RNA-Seq) and pipeline, in terms of their accuracy and precision. This talk will showcase preliminary results and highlight differences in workflow performances. A recommended workflow to maximize taxonomic profiling accuracy of microbial communities will also be presented.

scholarly journals RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

2019 ◽  
Author(s):  
Lin Di ◽  
Yusi Fu ◽  
Yue Sun ◽  
Jie Li ◽  
Lu Liu ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Reverse Transcription ◽  
Single Cells ◽  
Pcr Amplification ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
New Approach ◽  
Double Stranded Dna ◽  
Wide Range ◽  
Tn5 Transposase

AbstractTranscriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status but it relies on second strand cDNA synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol, and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.Significance StatementRNA sequencing is widely used to measure gene expression in biomedical research; therefore, improvements in the simplicity and accuracy of the technology are desirable. All existing RNA sequencing methods rely on the conversion of RNA into double-stranded DNA through reverse transcription followed by second strand synthesis. The latter step requires additional enzymes and purification, and introduces sequence-dependent bias. Here, we show that Tn5 transposase, which randomly binds and cuts double-stranded DNA, can directly fragment and prime the RNA/DNA heteroduplexes generated by reverse transcription. The primed fragments are then subject to PCR amplification. This provides a new approach for simple and accurate RNA characterization and quantification.

scholarly journals SLIDR and SLOPPR: Flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data

2020 ◽  
Author(s):  
Marius A. Wenzel ◽  
Berndt Mueller ◽  
Jonathan Pettitt
Keyword(s):  
Evolutionary Dynamics ◽  
Sequence Motifs ◽  
Rna Seq ◽  
Rna Molecules ◽  
Spliced Leader ◽  
C Elegans ◽  
Wide Range ◽  
Biological Insight ◽  
Bona Fide ◽  
Eukaryotic Genomes

AbstractBackgroundSpliced leader (SL) trans-splicing replaces the 5’ ends of pre-mRNAs with the spliced leader, an exon derived from a specialised non-coding RNA originating from a different genomic location. This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons have independently evolved multiple times throughout Eukarya, but our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes.ResultsHere we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5’ read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogation of sequence motifs expected in bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5’ SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that 1) SLIDR correctly identifies known SLs and often discovers novel SL variants; 2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons.ConclusionsSLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL trans-splicing and operons throughout Eukarya, and improve gene discovery and annotation for a wide-range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be derived from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.

scholarly journals SMARTer single cell total RNA sequencing

2018 ◽  
Author(s):  
Verboom Karen ◽  
Everaert Celine ◽  
Bolduc Nathalie ◽  
Livak J. Kenneth ◽  
Yigit Nurten ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Expression Patterns ◽  
Transcript Level ◽  
Cellular Heterogeneity ◽  
Circular Rnas ◽  
Rna Seq ◽  
Sequencing Data ◽  
Total Rna ◽  
Sequencing Experiment

AbstractSingle cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3’ end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.

Sign in / Sign up

Export Citation Format

Share Document