| Level | Description | Explanation |
|---|---|---|
| 1 | Multiple Direct Experimental Evidence | Identified and characterized experimentally by one direct method |
| 2 | Direct Experimental Evidence | Identified and characterized experimentally by one direct method |
| 3 | Inferred Based on indirect experimental evidence | Inferred from experimental data based on indirect method, e.g., mutation patterns in cDNA sequencing |
| 4 | Predicted Computationally | e.g., predicted from sequence, without experimental data |
| 5 | Unknown | Experimental evidence not yet annotated |
| 6 | Irrelevant | Experimental Evidence cannot be estimated for a given data type |
| Level | Description | Explanation |
|---|---|---|
| 1 | Rock Solid | It would be surprising to see it proven wrong |
| 2 | Solid | Large errors are unlikely, but cannot be completely excluded, minor errors should not undermine the meaning of the data |
| 3 | Speculative | More likely to be correct than incorrect, major errors may happen and minor errors are quite likely |
| 4 | Questionable | e.g., different results in publications and/or database entries |
| 5 | Unknown | Reliability assessment has not yet been performed |
| 6 | Irrelevant | Reliability cannot be estimated for a given data type |
Examples of direct/indirect methods used to detect and map modified residues. Direct methods analyze the RNA directly and provide clear data of a presence of a specific ribonucleotide residue with a particular chemical structure (A, U, C, G or a specific modified residue) at a particular position of the RNA chain. Indirect methods either do not analyze the RNA directly (e.g., require reverse transcription to DNA, and then analyze these DNA products) and/or they do not detect the actual modifications but rather sense some secondary effects of their presence and require additional assumptions to be fulfilled and/or use computational prediction from the experimental data.
| Method Name | General/Specific | Direct/Indirect | Method Type | Literature References |
|---|---|---|---|---|
| High-resolution X-ray crystallography | General | Direct | Biophysical | PUBMEDID: 31619810 |
| High-resolution NMR | General | Direct | Biophysical | PUBMEDID: 30997719 |
| High-resolution cryo-EM | General | Direct | Biophysical | PUBMEDID: 29143818 |
| MS/MS | General | Direct | Biophysical | PUBMEDID: 1706062 |
| RiboMethSeq | Specific (2’'-O-Me) | Indirect | Chemical probing + sequencing | PUBMEDID: 27302133 |
| ICE-Seq | Specific (I) | Indirect | Chemical treatment + sequencing | PUBMEDID: 25855956 |
| M1A-Seq | Specific (m1A) | Indirect | Immunoprecipitation + sequencing | PUBMEDID: 29072297 |
| ARM-Seq; DM-tRNASeq | Specific (m1A, m3C, m1G, m,2,2G) | Indirect | RNA de-modification + sequencing | PUBMEDID: 26237225 |
| meCLIP | Specific (m6A) | Indirect | Immunoprecipitation + sequencing | PUBMEDID: 33376190 |
| AlkAnilineSeq | Specific (m7G and m3C) | Indirect | Chemical probing + sequencing | PUBMEDID: 30370969 |
| JACUSA2 | Specific (Q) | Indirect | Biophysical + sequencing | PUBMEDID: 37811872 |
| HydraPsiSeq | Specific (Ψ) | Indirect | Chemical probing + sequencing | PUBMEDID: 32976574 |
| hmC-CATCH | Specific (hm5C) | Indirect | Chemical treatment + sequencing | PUBMEDID: 30278133 |
| HAC-Seq | Specific (m3C) | Indirect | Chemical treatment + sequencing | PUBMEDID: 33313824 |
| bisulfite RNA sequencing | Specific (m5C) | Indirect | Chemical treatment + sequencing | PUBMEDID: 19059995 |
| m7G-MaP-seq | Specific (m7G) | Indirect | Chemical treatment + sequencing | PUBMEDID: 31504776 |
| TRAC-seq | Specific (m7G) | Indirect | Chemical treatment + sequencing | PUBMEDID: 31619810 |