ArticlePloS one2015
Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 115 papers.
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Who cites it
115 citing papers in PubMed.
- Evaluating miRNA Sequencing and Integrative Proteomics for Identifying Plasma miRNAs as Potential Pharmacodynamic Biomarkers of IFNβ-1a Biologics.Clinical and translational science · 2025Trial
- OTTR-CLASH: improved biochemical and bioinformatic identification of Argonaute 2-mediated microRNA-target RNA interactions.bioRxiv : the preprint server for biology · 2026Article
- Network-based analysis reveals potential microRNA regulation of oncogenic pathways in SOX10-depleted uveal melanoma.Cellular and molecular life sciences : CMLS · 2026Article
- Gel-free library preparation for next-generation RNA sequencing and small RNA quantification.Communications biology · 2026Article
- 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
- Generation of high-quality single-stranded DNA for full-length and truncated genome standards of recombinant adeno-associated viruses.Molecular therapy. Methods & clinical development · 2025Article
- Optimizing Small RNA Sequencing for Salivary Biomarker Identification: A Comparative Study of Library Preparation Protocols.International journal of molecular sciences · 2025Article
- Review
- Absolute quantification of mammalian microRNAs for therapeutic RNA cleavage and detargeting.RNA (New York, N.Y.) · 2025Article
- Trinucleotide substrates under pH-freeze-thaw cycles enable open-ended exponential RNA replication by a polymerase ribozyme.Nature chemistry · 2025Article
- Validation of tRNA-derived fragments as diagnostic biomarkers in suspected acute stroke; limitations in analysis and quantification methods.Molecular therapy. Nucleic acids · 2025Article
- Sequencing technologies to measure translation in single cells.Nature reviews. Molecular cell biology · 2025Review
- Improved precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA sequencing.RNA (New York, N.Y.) · 2025Article
- Improved precision, sensitivity, and adaptability of Ordered Two-Template Relay cDNA library preparation for RNA sequencing.bioRxiv : the preprint server for biology · 2024Article
- Quantitative design of cell type-specific mRNA stability from microRNA expression data.bioRxiv : the preprint server for biology · 2024Article
- Involvement of MicroRNAs in the Hypersensitive Response of Capsicum Plants to the Capsicum Chlorosis Virus at Elevated Temperatures.Pathogens (Basel, Switzerland) · 2024Article
- Article
- SingmiR: a single-cell miRNA alignment and analysis tool.Nucleic acids research · 2024Article
- The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.RNA (New York, N.Y.) · 2024Article
55 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
High-throughput sequencing (HTS) has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA) and other small RNAs (sRNA). Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.
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