Table 1 Comparison of current m6A transcriptome-wide profiling techniques
Strategy | Technique | Principle | Resolution | Starting materials | Sensitivity | Specificity | Stoichiometry | Advantages | Limitations | References |
|---|---|---|---|---|---|---|---|---|---|---|
Antibody based | MeRIP-seq/m6A-seq | IP by m6A antibody; NGS | 100–200 nt | 2–400 μg mRNA | Medium | Medium | No | First method for global mapping of m6A, applicable, widely adopted; | Nonspecific antibody interactions; low resolution; without stoichiometry | |
PA-m6A-seq | IP by m6A antibody; photo-crosslinking; NGS | ~ 23 nt | 10 μg mRNA | Medium | Medium | No | Higher resolution | Live cells required, does not discriminate m6A from m6Am, nonspecific antibody interactions, without stoichiometry | [37] | |
m6A-CLIP/miCLIP | IP by m6A antibody; photo-crosslinking; NGS | 1 nt | 20 μg mRNA | High | High | No | Single-nucleotide resolution | Nonspecific antibody interactions, without stoichiometry | ||
m6A-LAIC-seq | Full-length RNA IP by m6A antibody; NGS | 1500 nt | 150 μg total RNA | Medium | Medium | Yes | Quantifying m6A stoichiometry | Does not differentiate adjacent m6A sites, low resolution | [39] | |
m6A-seq2 | m6A-IP on the pooled samples; NGS | 100–200 nt | 1.2/n μg mRNA per sample (n = total sample number) | Medium | Medium | No | Allows quantification across genes and samples; lower technical/batch variability, starting material, and costs | Nonspecific antibody interactions, low resolution, without stoichiometry | [40] | |
Chemical assisted | m6A-SEAL-seq | Methyl oxidation by FTO; NGS | 100–200 nt | 5 μg mRNA | High | High | No | Low input material, higher sensitivity | Low resolution, complicate procedures, without stoichiometry | [52] |
m6A-label-seq | Metabolic labeling of m6A site; NGS | 1 nt | 5 μg total RNA | Medium | Medium | No | Single-nucleotide resolution | Live cells required, does not discriminate m6A from m6Am, without stoichiometry | [29] | |
m6A-SAC-seq | MjDim1 treatment; NGS | 1 nt | 2–50 ng mRNA | High | low | N/A | Single-nucleotide resolution, quantitative tracking of m6A | Sequence preference, low specificity and efficiency | [53] | |
GLORI | Glyoxal and nitrite to deaminate unmethylated A to I; NGS | 1 nt | 100 ng mRNA | High | High | Yes | Single-nucleotide resolution, able to define m6A clusters, stoichiometric | High sequencing cost, extra procedures needed to discriminate m6A from some other A modifications | [46] | |
Enzyme assisted | DART-seq | Deamination adjacent to m6A modification targets; NGS | 1 nt | 10 ng to 1 μg total RNA | Low | Low | No | Single-nucleotide resolution, stoichiometric | Live cells required | [41] |
m6A-REF-seq | MazF RNase digestion; NGS | 1 nt | 100 ng mRNA | High | High | Yes (Only ACA) | High resolution, antibody free, stoichiometric | Only cover ACA motif | [43] | |
MAZTER-seq | MazF RNase digestion; NGS | 1 nt | 100 ng mRNA | High | High | Yes (Only ACA) | High resolution, antibody free, stoichiometric | Only cover ACA motif | [42] | |
eTAM-seq | Global A deamination by TadA, RT–PCR, and Sanger sequencing | 1 nt | 259 pg mRNA (10 cells) | High | High | Yes | Stoichiometric, much lower input requirement, deep-sequencing free | Require control transcriptome, less sensitive to sites of low methylation levels | [44] | |
TGS | PacBio SMRT | SMRT | 1 nt | N/A | Low | Low | Yes | Long-read sequencing on native RNA | Not been further developed or commercialized | [55] |
Nanopore DRS | Nanopore DRS | 1 nt | 500 ng to 1 μg mRNA | Low | Low | Yes | Long-read sequencing on native RNA, able to detect other modifications as well as m6A | High cost, low accuracy, high requirements of the quantity and quality of input RNA, require a low or no methylation control |