Table 2.

m1A mapping and measuring techniques

Techniques	Fundamentals	Advantages	Limitations
LC-MS	digestion to single nucleotide and UV detection of m1A based on its physicochemical properties	•quantitative method •standardized method •short test time	•no sequence context and localization information •inability to distinguish the source of m1A •not high enough sensitivity
m1A-ID-seq	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody; the enriched fragments were subjected to demethylase or mock treatment. Identification of the m1A sites within the peaks by using a truncation signature	•compared with Dimroth rearrangement, AlkB ensures RNA integrity as much as possible •low number of laborious steps	•inability to call m1A sites with single-nucleotide resolution •inability to call multiple m1A sites within peaks •E. coli AlkB does not erase all m1A sites
m1A-MeRIP-seq	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody. m1A causes both reverse transcription stops and mismatch generation, and m1A was converted to m6A by using the Dimroth rearrangement method, followed by analysis of the m1A sites according to the mismatch rate	•low number of laborious steps •improved resolution for m1A site recognition	•the alkaline conditions used for Dimroth rearrangement may affect RNA integrity •inability to call m1A sites with single-nucleotide resolution •Inability to call multiple m1A sites within peaks
m1A-seq	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody, TGIRT induced by RT at m1A sites, the remaining library building process is the same as m1A-MeRIP-seq	•TGIRT has higher processivity and mutation frequency at m1A sites •achieving single-base resolution •ability to call multiple m1A sites on RNA	•the alkaline conditions used for Dimroth rearrangement may affect RNA integrity •the lack of UMIs could lead to trouble in data analysis and mutation rate calculation
m1A-MAP	immunoprecipitation of m1A-enriched fragments using anti-m1A antibody, TGIRT/HIV RT-1306 induced by RT at m1A sites; the remaining library building process is the same as m1A-ID-seq	•achieved single-base resolution •the UMI used at the 3′-adaptor can eliminate repetition caused by PCR and improve the accuracy of detection •HIV RT-1306 has more powerful readthrough efficiency and higher mutation frequency	•TGIRT does not have sufficient readthrough activity for m1A-containing RNA fragments