Find positions varying in modification across isoforms with dmr isoform.
This command will compare the modification levels at common exonic positions for all isoforms of a gene.
Example
The input to dmr isoform is a single bedMethyl table that has been generated from a modBAM of reads aligned to a reference transcriptome (or transcriptome assembly).
This command also requires an accompanying GTF file with valid transcript models.
Below is an example command:
$ modkit dmr isoform \
${bedmethyl} \
isoform_dmr.bed \
--gtf ${gtf}
The bedMethyl table is expected to be bgzipped and have a tabix index.
To generate an isoform SVG plot like the one above, add the following arguments:
modkit dmr isoform \
${bedmethyl} \
isoform_dmr_${gene_name}.bed \
--plot path/to/plot/dir \
--gene-name ${gene_name} \
--gtf ${gtf}
Important:
--plotrequires either--gene-nameor--gene-idand cannot be used in whole-transcriptome mode (i.e. without one of those arguments). Attempting to use--plotwithout a gene specifier will result in an error.
Note that exons without marks in certain transcripts indicates that there is no data in the input bedMethyl.
The command modkit bedmethyl map-to-genome can map a transcript-aligned bedMethyl to genome coordinates.
Schema of output
| column | name | description | type |
|---|---|---|---|
| 1 | chrom | name of contig from GTF | str |
| 2 | chromStart | 0-based start position | int |
| 3 | chromEnd | 0-based exclusive end position | int |
| 4 | name | modification codes present at this position | str |
| 5 | score | likelihood ration score | float |
| 6 | strand | strand, ‘+’ or ‘-’ | str |
| 7 | p_value | p-value of alternative hypothesis | float |
| 8 | max_absolute_difference | largest effect size of all isoforms at this position | float |
| 9 | n_transcripts | number of transcripts (isoforms) contributing to this position | int |
| 10 | gene_id | gene-id from the GTF | str |
| 11 | gene_name | gene-name from the GTF or ‘-’ if not found | str |
| 12 | pooled_proportions | gene-level aggregate modification rate | str |
| 13 | per_isoform_proportions | JSON formatted string of per-transcript, per-modification proprotions | str |
| 14 | per_isoform_counts | JSON formatted string of per-transcript, per-modification counts | str |
Columns 12 and 14 are only present when the --full flag is passed.
Background
Gene sequences alone don’t describe all of the diversity of mRNAs in vertebrate cells. Through alternative splicing gene exons can be combined in multiple permutations called isoforms.
Filtering the number of methylation marks
Some long genes may have many modified positions and drawing them all can get crowded.
To only plot positions with a maximum p-value or minimum score use the --max-pval or
--min-score arguments, respectively.
Note:
--max-pvaland--min-scoreare plot-only options. They require--plot(and therefore also--gene-nameor--gene-id) and have no effect outside of single-gene mode. These two flags are mutually exclusive — only one may be provided at a time.