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- W3159597439 abstract "Abstract Terminal nucleotidyl transferases are enzymes that add non-templated nucleotides to RNA molecules. In the case of microRNAs, this process was shown to be functionally relevant for their maturation process and generation of isomiRs with non-canonical mRNA targets. Deconvolution of these posttranscriptional modifications is challenging in particular for extracellular miRNAs that are considered as a target for minimally-invasive diagnostics. Massively parallel RNA sequencing is the only method that can truthfully reveal isomiR diversity in biological samples and determine relative quantities. Improvements aside, current small RNA sequencing strategies remain imprecise. We developed IsoSeek that diverges from these methods by making use of randomized 5’- and 3’-adapters combined with a 10N unique molecular identifier (UMI). Using synthetic miRNA and isomiR spike-in sets and testing depletion and RNA competition strategies in 7 sequencing rounds of >100 samples, we rigorously optimized and validated the technical accuracy of the IsoSeek method. In genetically-altered HEK293, we characterized the terminal uridylase (TUT4/TUT7) dependent miRNA uridylome and discovered extensive uridylation of disease-associated miRNAs. Notably, 3’-uridylated isomiR profiles of plasma extracellular vesicles (EVs) rely on UMI-correction. Thus, IsoSeek advances our knowledge of cell-free miRNAs and supports development into non-invasive biomarkers." @default.
- W3159597439 created "2021-05-10" @default.
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- W3159597439 date "2021-05-04" @default.
- W3159597439 modified "2023-10-10" @default.
- W3159597439 title "Unbiased and UMI-informed sequencing of cell-free miRNAs at single-nucleotide resolution" @default.
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- W3159597439 doi "https://doi.org/10.1101/2021.05.04.442244" @default.
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