Modification mapping and site-level profiling
Where a modification sits, and how much of it is there. This is the question the single-base line answers directly, mark by mark, one nucleotide at a time.
01The question
Site-level mapping asks two things at once: which transcripts carry a mark, and where inside them the modified nucleotide sits. A map is only useful if it can answer both, which is why the position has to be resolved rather than inferred from an enriched region.
The distinction matters because the two commonly used routes do not answer the same question. Enrichment-based profiling reports where signal accumulates across a transcript; single-base chemistry reports the individual nucleotide and the fraction of transcripts modified at it.
A mapping study therefore starts from the resolution the conclusion needs. If the result is going to name a position, the route has to produce one.
02Which route produces a map
Every mark with a single-base method is listed on the modification types page; the four below are the most common starting points.
m6A Modification Seq (CAM-seq)
Chemical deamination of unmodified adenosine leaves m6A readable at base resolution, with a ratio at each site.
Site-levelposition & level m5Cm5C Modification Seq (BS-seq)
Bisulfite conversion distinguishes modified from unmodified cytosine position by position.
Site-levelposition ALL MARKSSingle-Base RNA Modification Sequencing
Sixteen methods across m6A, m5C, m1A, ac4C, m7G, m3C, Ψ, inosine, 2′-O-Me, D and O8G.
16 methodsone line REFERENCEModification types
Which marks can be resolved to a base, and which are covered only by enrichment.
Reference13 marks03What a mapping result contains
- Site tables. Genomic coordinate, host gene, functional region and modification ratio for every called site.
- Motif and distribution. Consensus motif logos and transcript feature distributions, so the map can be compared with published sets.
- Differential modification. Site-level comparison between conditions or sample groups, with fold change and significance.
- Track files. Genome-browser compatible signal so the map can be inspected alongside other datasets.
- Raw data and QC. Sequencing files formatted for GEO/SRA submission, plus the alignment and mapping statistics behind the calls.
04Setting up a mapping project
- Fix the mark and the material: which modification, and total RNA, cells, tissue or blood.
- Decide the comparison. A map on its own describes one state; a mapping study usually compares two.
- Confirm the resolution. If the conclusion names a position, the route has to be a single-base one.
- Run the workflow, then read the site table against the question rather than the other way round.
| What you want to conclude | Route | Request Quote |
|---|---|---|
| A specific nucleotide is modified, at a given level | Single-base sequencing | |
| A transcript or region carries signal | RIP-sequencing | |
| Which of many targets changed | Modification array |
05FAQ
Can an enrichment-based profile be used as a map?
No. Enrichment reports where signal accumulates across a transcript, not which nucleotide carries the mark, so a peak is a candidate region rather than a coordinate. It is a good way to choose targets, and the position then comes from a single-base method run on the same material.
What does the modification ratio at a site mean?
It is the fraction of transcripts in the sample that carry the modification at that position, estimated from the read composition at the site. Because it is a property of the sample rather than of the transcript, it is the value to compare between conditions, and it does not by itself say what the mark does.
Do I need a comparison to make a map useful?
Not always, but most mapping studies compare two states, because a single map describes one condition and cannot separate a real difference from sample-to-sample variation. The interesting result is usually the difference between two, so the comparison is worth fixing before the samples are run. A map on its own is a description, not a finding.
How do I know which marks can be mapped this way?
The modification types page lists all thirteen marks and states which have a single-base method and which are covered only by enrichment. Where a mark has no single-base route, no sequencing method can return its position, so the study is designed around a region-level readout from the start.