Two ways to read m6A at base resolution: CAM-seq and GLORI2-seq
Both routes convert unmodified adenosine and leave m6A standing, so both read position and level from the same A-to-G signal. What separates them is the chemistry around that conversion.
01What this article answers
CAM-seq and GLORI2-seq are antibody-free chemical methods for m6A. Each deaminates unmodified adenosine to inosine, which is copied as guanosine, while m6A resists the reaction and is copied as adenosine. The A/G balance at a position therefore reports both where the modification sits and how much of that position carries it.
Because the two methods reach the same readout through different chemistry, the practical question is not which signal is better but which reaction conditions suit the material at hand. Choose by the reaction conditions your material can take.
Neither route relies on an antibody, and neither depends on a canonical motif to find a site.
02Where the two routes differ
The two chemistries share the logic — convert the unmodified base, leave the modification intact — and differ in how that conversion is driven and in what has to be protected while it runs.
| CAM-seq | GLORI2-seq | |
|---|---|---|
| Conversion chemistry | Cooperative catalysis: a carbonyl organocatalyst works together with boric acid | One-pot reaction of glyoxal, boric acid and sodium nitrite |
| Reaction conditions | Mild, near-neutral pH | Single-pot conversion, without a dedicated guanosine-protection step |
| RNA protection | A reversible kethoxal group protects guanine through the treatment | RNA stays intact through treatment and library construction without a separate protection step |
| Workflow shape | Chemical treatment after sample QC, then library construction | RNA fragmentation, one-pot treatment, then library construction |
| Readout | A-to-G signature gives the site and its modification ratio | A/G ratio at a position gives absolute m6A stoichiometry |
| Transcript scopes | mRNA, or mRNA together with lncRNA | mRNA, or mRNA together with lncRNA |
The protection step is the clearest difference. CAM-seq carries a reversible kethoxal group that keeps guanine protected while the catalyst works, which is what allows the mild, near-neutral conditions. GLORI2-seq removes that step altogether: the conversion runs in one pot, which leaves fewer handling steps between the RNA sample and the library.
Earlier protection-based deamination chemistry was the reason this step mattered, because it was harsh on RNA and pushed input requirements up. CAM-seq addresses that with a milder protecting group, and GLORI2-seq by doing without one.
03How to choose
Both services are offered in the same two transcript scopes and both end in a site-level table with stoichiometry, so the choice comes down to the condition differences above.
- How much material can the study spare? The route that fits best is the one whose chemistry and handling demands sit inside what the sample can support.
- How much pressure is on RNA integrity? Both routes are built to keep transcripts intact; GLORI2-seq does so without a separate protection step, which removes handling between treatment and library construction.
- Which chemistry does the group want to build on? Contributions that use cooperative catalysis and a kethoxal protecting group are read alongside contributions that use the one-pot glyoxal route; both are deamination chemistries, and the data they produce is read the same way.
- Is the same chemistry needed on another mark? Where the project extends to pseudouridine or another mark, the other single-base services are chosen on the same principle: the chemistry that fits the sample.
04Limits and common misreadings
- This article does not rank the two methods. They differ in reaction conditions, not in ambition, and neither is presented here as the better route.
- Both read a conversion signature. The site call comes from the A-to-G signal, so the interpretation of a position and its ratio is shared between the two methods.
- Neither route reports enrichment. An A/G ratio is not an enrichment score, and a value from these services should not be compared with a peak intensity from an antibody-based profile.
- The cap is a separate question. m6Am at the cap-proximal position is read on the RIP line, not by these two methods.
06FAQ
Do CAM-seq and GLORI2-seq report the same quantity?
Yes, both report a modification level at each called site, read from the balance between converted and unconverted reads at that position. The chemistry behind the conversion differs, which is what the table above sets out, so the number is read the same way even where the route to it is not.
Does either method use an antibody?
No. Both are antibody-free: detection depends on the chemistry acting on unmodified adenosine, not on a capture step. Neither route relies on a canonical motif to find a site either, so the site list comes from the conversion itself rather than from where an antibody binds.
Which of the two should be chosen for a low-input project?
That is a question about the sample rather than about the mark, so it is settled by the material the study can supply. The route worth discussing with technical support is the one whose reaction conditions and handling sit inside what that material supports, which is where the removal of a separate protection step matters.
Can lncRNA be covered in the same project?
Yes, on either route. Both offer two transcript scopes: mRNA alone, or mRNA together with long non-coding RNA, analyzed within one experiment. That scope is set when the project is planned, so a study that may extend to lncRNA later is best designed with the scope from the start.