TABLE 2.

Methods for detecting RNA modifications.

Methods	Detection	Pros	Strengths	Drawbacks
Direct sequencing	Via nucleotide labeling and chromatography	Various modifications	● Powerful and able to produce high quality data	● Labor intensive ● Restricting its use to highly abundant RNAs
Direct sequencing with SCARLET	Site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography	m6A Ψ	● Purifing less abundant species of RNA ● Including sequence information ● Quantification possible ● No specialized equipment	● Single-site query ● No high throughput ● Labor intensive
Direct sequencing with mass spectrometry	Analyze fragmented and whole RNA	Various modifications	● Unbiased manner ● Highly accurate quantification ● Expertise is reasonably widespread	● Labor-intensive ● No sequence information ● Requires specialized equipment ● Methodological and computational challenges
Reverse transcriptase-based methods	Detecting the stalling and termination	Various modifications	● Targeting transcripts in a heterogeneous pool of RNA ● Ideal approach for studying less abundant RNAs ● Straightforward protocol ● Precise single-nucleotide mapping ● Adaptable to different types of modification	● Semi-quantitative
High-resolution melting	DNA polymorphisms DNA methylation covalent RNA modifications	Various modifications	● Performing with any existing set of PCR probes ● Covering a putative modification site	● Putative modifications ● Relying on a shift in melting temperature.
Global methods for detecting RNA modifications
Antibody-based enrichment coupled to high-throughput sequencing	Methyl and hydroxymethyl RIP-seq, which rely upon antibodies recognizing modified ribonucleotide epitopes	m6A m1A Ψ m5C hm5C	● Unbiased surveys	● Modification sites cannot be defined with singlenucleotide resolution
High-throughput sequencing with chemical-based methods	Specifically target or exclude modified ribonucleotides with high-throughput sequencing	m6A m5C Ψ	● Determining the location of modification sites ● Single-nucleotide resolution techniques	● Potential false negatives ● Apparent mismatches from the expected sequence
High-throughput single-molecule sequencing	Directly measuring changes in base pairing like SMRT and Nanopore sequencing	m6A, m5C, hm5C Inosine Ψ	● Much longer sequencing-length ● Allow direct readout of modification sites ● Providing unbiased views of both the transcriptome and epitranscriptome ● Allowing direct quantitation of modification abundance	● Prone to noise and sequencing error ● Statistics problems ● Unmatured base-calling
In silico methods	High-throughput analysis of modified ribonucleotides	Various modifications	● Identifying modifications transcriptome-wide with single nucleotide resolution ● Retrospectively and can be readily applied to existing data and in meta analyses. ● Surveying multiple ● modification subtypes simultaneously	● Artifacts not be properly controlled Multiplesteps ● Limited to diploid and haploid organisms

m⁶A, N⁶-methyladenosine; m⁵C, 5-methylcytidine; m1A, N1-methyladenosine; Ψ, pseudouridine; hm5C, 5-hydroxymethylcytosine; SCARLET, site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography; SMRT, single molecule real-time; snRNAs; small nuclear RNAs; snoRNAs, small nucleolarRNAs.