Ligation bias is a major contributor to nonstoichiometric abundances of secondary siRNAs and impacts analyses of microRNAs

Baldrich, Patricia; Tamim, Saleh; Mathioni, Sandra M.

doi:10.1101/2020.09.14.296616

Cited by 5 publications

(4 citation statements)

References 35 publications

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“…Specifically, the miR845 family increased until occupying close to 45% of the all sRNAs at pollen maturity ( Figure 1D ). Although this relative enrichment could be due to a bias in sRNA sequencing ( Baldrich et al, 2020 ), we found a consistent and significant detection of this increase in trinuclear and mature pollen sRNA libraries ( Figure 1, D, E , and I ). We confirmed this accumulation trend by sRNA gel blot for miR845, miR156, and miR161.1, which increased, maintained, and decreased their accumulation level respectively ( Figure 1F; Supplemental Figure S1, D and E ).…”

Section: Resultssupporting

confidence: 59%

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

et al. 2021

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Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity.

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Section: Resultssupporting

confidence: 59%

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

et al. 2021

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“…In our hands, NEB was the protocol that showed the highest reproducibility between replicas and that resulted in the highest number and variety of miRNAs, followed by NEXT and SMARTer. Our results are consistent with other studies [41][42][43] and reinforce the choice of NEB over other protocols because it is an easy-handling procedure specially applicable to low-input material, as is the case of clinical samples. As a second option, and depending on the source of the input material, the NEXT protocol might be the library preparation of choice [25,44].…”

Section: Discussionsupporting

confidence: 92%

Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

Llorens-Revull

Martínez-González

Quer

et al. 2023

IJMS

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MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.

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“…However, it must be assumed that this is not the case when comparing between liquid biopsy sample types with very specific RNA compositions [19] or between different individuals [34]. Lutzmayer et al recognized this need in the context of plant microRNA research [33,35], but the adaptation of the technology to mammalian species as well as liquid biopsies was still missing.…”

Section: The Value Of Spike-ins For Small Rna Sequencingmentioning

confidence: 99%

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

Khamina¹,

Diendorfer²,

Skalicky³

et al. 2022

IJMS

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The plasma levels of tissue-specific microRNAs can be used as diagnostic, disease severity and prognostic biomarkers for chronic and acute diseases and drug-induced injury. Thereby, the combination of diverse microRNAs into biomarker signatures using multivariate statistics seems especially powerful from the perspective of tissue and condition specific microRNA shedding into the plasma. Although next-generation sequencing (NGS) technology enables one to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases (e.g., adapter ligation bias) and lacks absolute transcript quantitation as well as tailor-made quality controls. 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 quality control and absolute quantitation, thus ensuring 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 biofluids in the setting of any diagnostic, prognostic or patient stratification need. The established miND assay was tested on serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture medium of primary cells and proved its potential to be used across different sample types.

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Ligation bias is a major contributor to nonstoichiometric abundances of secondary siRNAs and impacts analyses of microRNAs

Cited by 5 publications

References 35 publications

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

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

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