Reanalyses of sequencing data by Safra et al. (2017) provide an explanation as to why they failed to identify the majority of m1A in mRNA. (A) Reads’ coverage for the eight TRMT6/61A sites (detected in both studies) in different sequencing data sets. The inset box in each panel provides a zoomed-in view of reads’ coverage by Safra and coworkers. (B) Reads’ coverage for the 44 TRMT6/61A sites and 196 “reclassified” TSS sites in different sequencing data sets. All these sites are missed in the study by Safra and coworkers. The inset box in each panel provides a zoomed-in view of reads’ coverage by Safra et al. (2017). (C) Reads’ duplication levels and rRNA contamination levels of different sequencing data sets. (D) Mismatch rates in the “IP” and “IP + demethylation” samples for m1A1322 of 28S rRNA and m1A947 of 16S mt-rRNA. (E) Mismatch rate in the “IP” and “IP + demethylation” samples for a novel m1A575 site of 12S mt-rRNA. This site is biochemically validated by Li et al. (2017) but missed by Safra et al. (2017). (F) IGV views for the raw sequencing data and misincorporations for m1A575 site of 12S mt-rRNA. (G) Reanalysis of the SSIII data by Safra et al. (2017) showing poor data quality that does not even support their own TGIRT data. The analysis was performed by aligning the reads to the genome reference using TopHat2. (H) IGV views for one modification site that is claimed as false positive due to its coincidence with SNP by Schwartz (2018). (I) IGV views for one modification site that is claimed as false positive due to its location within a polyC stretch.
