Unbiased and UMI-informed sequencing of cell-free miRNAs at single-nucleotide resolution

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.

Reassessment of miRNA variant (isomiRs) composition by small RNA sequencing

“… 8 We first scrutinized this claim by analyzing the canonical miRNA and isomiR profiles from 10 different protocols generated furthermore by different laboratories. Specifically, we analyzed data from a synthetic 963-miRNA reference pool (miRXplore Universal, Miltenyi Biotec) and HEK293T cellular RNA using commercial and non-commercial randomized-end adapter protocols (AQ-seq, 6 4N-G and 4N-X, 5 , 6 NEXTflex, and our own 5N “IsoSeek” method 7 ), one custom 2N adapter protocol (AQRNA-seq 8 ), and three commercial invariant (fixed) adapter protocols (NEBNext, 13 , 19 TruSeq, 20 QIAseq, 21 , 22 and Clean-Tag 22 ). The main characteristics of the different protocols are summarized in Table S1 .…”

“…The 5′- and -3′′-adapter sequences are based on the adapters from the NEBNext Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs) with the addition of 5 random nucleotides (5N) as described by van Eijndhoven et al. 7 …”

“…All small RNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina with fixed NEBNext adapters from the kit or our custom designed 5′- and -3′′-5N-adapters (IsoSeek). 7 …”

“… 4 , 5 To overcome this, recent sequencing protocols make use of random nucleotides at one or both sequencing adapters, thereby reducing detection bias of canonical miRNAs. 5 , 6 , 7 , 8 However, accurate detection of post-transcriptional modifications remains challenging, in particular for low-input applications such as extracellular miRNA profiling used for minimally invasive diagnostics.…”

“…To avoid any bioinformatics processing bias, all sample data were uniformly processed using sRNAbench, 17 a broadly used miRNA analysis software that can also accommodate virtually any sequencing protocol. We also included samples from “IsoSeek,” our thoroughly optimized 7 in-house 5N protocol that aims to account for all technical bias by using 5′ and 3′ randomized adapters, each of them with 5 random nucleotide unique molecular identifiers (UMIs) (5N) based on commercial adapters. 18 Our results show that regardless of the adapter strategy, the majority of protocols generate only minor library preparation artifacts and detect biological variants with relevant overlap, mostly 3′ end NTAs.…”