Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Systematic assessment of next-generation sequencing for quantitative small RNA profiling: A-to-I editing pools

ABSTRACT: Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed reproducibility and accuracy of relative expression measurements using two pools of synthetic small RNA sequences, where subsets of the sRNAs vary in relative amount between pools A and B. The pools each contain 334 small RNAs, varying by 15 different ratios between pools A and B (10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 1:5, 1:8, 1:10). We find that although the sequencing bias varies extensively between protocols, the relative abundance measured between samples A and B is largely reproducible and accurate across labs and protocols. These results suggest that measurements of differential expression should be comparable across institutions and library preparation technologies.

ORGANISM(S): synthetic construct

PROVIDER: GSE108138 | GEO | 2018/07/09

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Systematic assessment of next-generation sequencing for quantitative small RNA profiling: synthetic ratiometric pools

Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed reproducibility and accuracy of relative expression measurements using two pools of synthetic small RNA sequences, where subsets of the sRNAs vary in relative amount between pools A and B. The pools each contain 334 small RNAs, varying by 15 different ratios between pools A and B (10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4 1:5, 1:8, 1:10). We find that although the sequencing bias varies extensively between protocols, the relative abundance measured between samples A and B is largely reproducible and accurate across labs and protocols. These results suggest that measurements of differential expression should be comparable across institutions and library preparation technologies.

2018-07-09 | GSE94585 | GEO

Systematic assessment of next-generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories

Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols. This SuperSeries is composed of the SubSeries listed below.

2018-07-09 | GSE94586 | GEO

Systematic assessment of next-generation sequencing for quantitative small RNA profiling: synthetic equimolar pool

Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed sequencing bias and reproducibility using an equimolar pool of 1,152 small RNA sequences ranging from 15-90 nt, and primarily comprised of annotated human microRNAs. We observed extensive protocol-specific and sequence-specific bias that was largely mitigated in protocols employing sequencing adapters with randomized end-nucleotides. We find that sequencing bias is highly reproducible across labs using the same library preparation technologies, and use the data to calculate inter-protocol bias correction factors. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

2018-07-09 | GSE94584 | GEO

Systematic assessment of next-generation sequencing for quantitative small RNA profiling: human plasma pool

Project description:Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. Using common sets of reference samples, we evaluated the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. As part of this larger study, we assessed reproducibility and diversity of sequencing of mature miRNAs, generating libraries from RNA extracted from human plasma that was pooled from 11 healthy individuals. We find that protocol-specific biases are largely recapitulated in the biological samples, and that inter-protocol bias correction factors estimated from the more comprehensive equimolar synthetic pools can be applied to the biological samples to get more comparable, and in some cases, more accurate estimates of miRNA abundance.

2018-07-09 | GSE94582 | GEO

Evaluating bias-reducing protocols for RNA sequencing library preparation

Project description:Evaluating bias-reducing protocols for RNA sequencing library preparation

| PRJEB6271 | ENA

A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) For Genome-Scale and Absolute Quantitation of Circulating MicroRNA Biomarkers

Project description:The plasma levels of tissue-specific microRNAs can be used as prognostic and diagnostic biomarkers for chronic and acute diseases. Thereby, the combination of diverse miRNAs into biomarker signatures using multivariate statistics seems especially powerful in view to tissue and condition specific miRNA shedding into the plasma. Although Next-Generation Sequencing (NGS) technology enables to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases (e.g. adapter ligation bias) and lacks absolute transcript quantitation. In order to develop a robust NGS discovery assay for genome-scale quantitation of circulating microRNAs we first evaluated the sensitivity, repeatability and ligation bias of four commercially available small RNA library preparation protocols. The protocol from RealSeq Biosciences was selected based on its performance and usability, and coupled with a novel panel of exogenous small RNA spike-in controls to enable absolute quantitation and ensure comparability of data across independent NGS experiments. The established MicroRNA Next-Generation-Sequencing Discovery Assay (miND) was validated for its relative accuracy, precision, analytical measurement range and sequencing bias and was considered fit-for-purpose for microRNA biomarker discovery. Summarized, all these criteria were met and thus our analytical platform is considered fit-for-purpose for microRNA biomarker discovery from plasma, serum, cerebrospinal fluid (CSF), synovial fluid (SF), or extracellular vesicles (EV) extracted from cell culture medium in the setting of any diagnostic, prognostic or patient stratification need.

2022-01-20 | GSE189930 | GEO

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

Project description:We performed a parallel analysis of commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se generates pronounced sequencing biases, and subsequent fragmentation and amplification steps lead to several-fold overrepresentation of these artefacts. Standard pre-bisulfite library preparation methods lead to a significantly biased genomic sequence representation and a marked overestimation of methylation levels. We have integrated a bias diagnostic tool in the Bismark package and propose that amplification-free and post-bisulfite procedures should become the gold standard for WGBS library preparation.

2017-02-16 | GSE77961 | GEO

Project description:Systematic comparison of small RNA library preparation protocols for next-generation sequencing

| PRJNA429731 | ENA

Project description:Systematic comparison of small RNA library preparation protocols for next-generation sequencing

| PRJNA429634 | ENA

Performance comparison of small RNA-Seq library preparation methods for biofluid samples

Project description:We report performance of six different protocols for small RNAseq library preparation and of a method utilizing sequencing of probes targeting microRNAs (HTG EdgeSeq). Recently, small RNA sequencing (small RNA-seq) has been introduced as a method for quantifying circulating microRNAs (miRNAs) and enabling their global profiling without prior knowledge of target sequences. Despite its great promise, small RNA-seq has not delivered the expected outcomes, particularly due to ligation and PCR bias introduced within the workflow. In this study, we assessed the performance of all existing approaches to the small RNA-seq of miRNAs in plasma samples: original two adapter ligation approach; single adapter ligation with subsequent circularization; polyadenylation; use of randomized adapters; and use of unique molecular identifiers (UMI). Using comprehensive set of metrics, we evaluated each protocol in terms of yield, precision, accuracy, sensitivity, and ability to detect isomiRs. Moreover, we assessed performance of targeted RNA-seq method utilizing hybridization probes across relevant metrics and together with RT-qPCR we used it as a reference for accuracy evaluation. The best results were delivered by targeted RNA-seq outperforming other methods in all relevant parameters. The protocols using randomized adapters or UMIs showed consistent good performance across all of the assessed metrics. In contrast, the polyadenylation approach generated a high percentage of discarded reads and impeded the analysis of isomiRs. The single adapter ligation with subsequent circularization failed to prevent ligation bias and the traditional two adapter ligation approach achieved the worse scores in the majority of tested metrics. To sum, we provide a comprehensive comparison that can serve as a guide for new users interested in analysis of circulating miRNAs and as a reference for further comparative studies.

2020-12-31 | GSE149513 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data