The analytical method described in Mai et al. (2020) is not specific for piR-54265. (A) Schematic diagram of a C/D box snoRNA (black), with its associated proteins and pre-rRNA (red). (B) Schematic representation of the stem–loop RT-ddPCR strategy designed in Mai et al. (2020) for the analysis of piR-54265 (top), also amplifying full-length SNORD57 (bottom). (C) Genome browser coverage tracks of TGIRT-seq reads obtained in plasma from a healthy human subject. (Bottom panel) Read coverage in the SNORD57 gene, showing no support for circulating piR-54265. (D) Illustration of the problem of identifying piRNAs based on mapping small RNA-seq data to piRNA databases, which contain entries that correspond to ncRNA fragments (shown in red) as well as bona fide piRNAs (shown in gray).
