ArticleBMC genomics2018
Systematic comparison of small RNA library preparation protocols for next-generation sequencing.
Article in BMC genomics, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 75 papers.
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Who cites it
75 citing papers in PubMed, 128 citations in OpenAlex.
- Non-Coding RNA Biomarkers in Male Infertility: From Discovery to Clinical Actionability-A Narrative Review.Genes · 2026Review
- An absolute quantification atlas of small non-coding RNAs across diverse mammalian tissues and cell lines.Nature communications · 2026Article
- Micro RNA Sequencing in Circulating Tumor Cells.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Designing RNA sequencing experiments: A practical guide to reproducible gene expression analysis.Computational and structural biotechnology journal · 2026Review
- Exploring microRNAs, One Cell at a Time.Non-coding RNA · 2025Article
- New approaches in the analysis of spent embryo culture media in the IVF process.Archives of gynecology and obstetrics · 2025Article
- Small Nucleolar RNAs: Biological Functions and Diseases.MedComm · 2025Review
- Full sequencing of 100mer sgRNA via tandem mass spectrometry by targeted RNase H digestion with customized probes.Analytical and bioanalytical chemistry · 2025Article
- A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.Molecular cell · 2024Article
- Guidelines for mitochondrial RNA analysis.Molecular therapy. Nucleic acids · 2024Review
- SingmiR: a single-cell miRNA alignment and analysis tool.Nucleic acids research · 2024Article
- Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.Nucleic acids research · 2024Article
- Single-Cell Transcriptome Sequencing Reveals Molecular Expression Differences and Marker Genes in Testes during the Sexual Maturation of Mongolian Horses.Animals : an open access journal from MDPI · 2024Article
- Small RNA structural biochemistry in a post-sequencing era.Nature protocols · 2024Review
- Single Cell Micro RNA Sequencing Library Preparation.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing.International journal of molecular sciences · 2023Article
- Urinary phthalate metabolites and small non-coding RNAs from seminal plasma extracellular vesicles among men undergoing infertility treatment.Environmental pollution (Barking, Essex : 1987) · 2023Article
- A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.bioRxiv : the preprint server for biology · 2023Article
- Adapter dimer contamination in sRNA-sequencing datasets predicts sequencing failure and batch effects and hampers extracellular vesicle-sRNA analysis.Journal of extracellular biology · 2023Article
- Small RNAs and Karma methylation in Elaeis guineensis mother palms are linked to high clonal mantling.Plant molecular biology · 2023Article
15 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundNext-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This inhibits 3' adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina's classical TruSeq protocol regarding the detection of normal and 2' OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance.
resultsAmong the five kits tested with their respective standard protocols, the SMARTer and CATS kits had the lowest levels of bias but also had a strong formation of side products, and as a result performed relatively poorly with biological samples; NEXTflex detected the largest numbers of different miRNAs. The use of a novel type of randomised adapters called MidRand-Like (MRL) adapters and PEG improved the detection of 2' OMe RNAs both in the TruSeq as well as in the NEXTflex protocol.
conclusionsWhile it is commonly accepted that biases in sRNA library preparation protocols are mainly due to adapter ligation steps, the ligation-free protocols were not the best performing methods. Our modified versions of the TruSeq and NEXTflex protocols provide an improved tool for the study of 2' OMe RNAs.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.