RT Journal Article
SR Electronic
T1 Unbiased and UMI-informed sequencing of cell-free miRNAs at single-nucleotide resolution
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.05.04.442244
DO 10.1101/2021.05.04.442244
A1 M.A.J. van Eijndhoven
A1 E. Aparicio-Puerta
A1 C. Gómez-Martín
A1 J.M. Medina
A1 E.E.E. Drees
A1 E.J. Bradley
A1 L. Bosch
A1 C. Scheepbouwer
A1 M. Hackenberg
A1 D.M. Pegtel
YR 2021
UL http://biorxiv.org/content/early/2021/05/04/2021.05.04.442244.abstract
AB 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.Competing Interest StatementD.M.P. and M.H. are co-founders of Exbiome BV. D.M.P. is CSO of ExBiome BV and served as an advisor for Takeda for which he received travel compensation.