PRJNA867189
Reassessment of miRNA variant (isomiR) composition by small RNA sequencing
Minor sequence variants (isomiRs) generated by post-transcriptional modification of miRNAs are thought to diversify the miRNA targetome. Library preparation introduce artifacts and bias and to avoid that current protocols employ randomized sequence adapters. How these compare to classic protocols in terms of isomiR calling remains unclear. We performed a comparative analysis of 8 small RNA-seq protocols, exploring data from a, theoretically, isomiR-free pool of synthetic miRNAs and HEK293T cells. We calculated that less than 5% of miRNA reads can be attributed to library-preparation artifacts except for two protocols. Moreover in the cells we detect higher percentages of biologically relevant isomiRs. We also show considerable accuracy and concordance between protocols in detecting NTA-U isomiRs using terminal-RNA uridyl transferases knock-out cells. Our data reveals that 6 protocols generally accurately detect true isomiRs while 2 suffer from false isomiR generation, which is relevant for understanding the physiological role of isomiRs and their biomarker potential.