Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen), as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq), or unique molecular identifiers (QIAseq). There was no single protocol outperforming others across all metrics. Limited overlap of measured miRNA profiles was documented between methods largely owing to protocol-specific biases. Methods designed to minimize bias largely differ in their performance, and contributing factors were identified. Usage of unique molecular identifiers has rather negligible effect and, if designed incorrectly, can even introduce spurious results. Together, these results identify strengths and weaknesses of all current methods and provide guidelines for applications of small RNA-Seq in biomarker research.

Laboratory of Gene Expression Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czech Republic

Laboratory of Gene Expression Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czech Republic; Department of Anthropology and Human Genetics Faculty of Science Charles University Prague Czech Republic

Laboratory of Gene Expression Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czech Republic; Department of Informatics and Chemistry Faculty of Chemical Technology University of Chemistry and Technology Prague Czech Republic

Laboratory of Gene Expression Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czech Republic; Laboratory of Growth Regulators Faculty of Science Palacky University Olomouc Czech Republic

Laboratory of Gene Expression Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czech Republic; TATAA Biocenter AB Gothenburg Sweden

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis