Methylation & imprinting

Long-read sequencing reads DNA methylation directly from the same data used for variant calling, with no separate assay. Because reads are phased, methylation can be assigned to each parental copy, which is what makes imprinting analysis possible.

Imprinting is when a gene is normally expressed from only one parental copy. Loss of imprinting, the expected pattern being disrupted, underlies several imprinting disorders. The Methylation tab lists the sample's methylation calls over imprinted, differentially methylated regions, so you can find regions whose methylation departs from what you'd expect.

The Methylation tab appears only when the case includes methylation results for the sample.

The methylation table

Each row is one region. The default columns include Locus, Overlapping Genes, the region's coordinates, Mean Coverage, Mean Mod Score (the primary methylation measure, from 0 to 100), Source, Cytogenetic Band, CpGs Observed, Pooled Meth %, Fraction Sites Meth, and Meth Threshold. Rows are sorted by chromosome, then start position.

Source tells you which reads the values come from: Combined (both haplotypes together), Haplotype 1, or Haplotype 2.

Filters

The filter drawer offers Mean Mod Score, Source, Mean Coverage, CpGs Observed, Chromosome, and Start Position.

The tab opens pre-filtered to regions with a Mean Mod Score of 15 or below, or 85 or above, from the Combined source. These are the strongly hypomethylated and hypermethylated regions. Clear or widen these filters to see every region.

How methylation findings are flagged

A region is flagged when its combined mean modification score is 15 or below (Hypomethylated) or 85 or above (Hypermethylated). These regions appear as priority findings on the Summary tab, in the Weak evidence tier, marked as hypomethylated or hypermethylated.

Flagging uses a fixed cutoff, not a comparison with a population reference range for each region. No expected range per locus is loaded, so the detail view can't show one. Judge whether a flagged region is abnormal for its locus from your own knowledge of the expected imprinting pattern.

The region detail

Opening a region shows the Variant Summary, Region composition, and Methylation cards. A Not available for this variant type card lists the evidence methylation calls don't carry yet:

• Expected methylation range: no expected range per locus is ingested.

• Per-allele split: in the detail view, methylation is pooled across both haplotypes. To compare haplotypes, use the Source filter in the table.

• Clinical evidence: there's no disease annotation for imprinted loci.

• Predicted effect: there's no automated classification of methylation calls.

The Browser tab opens a family view with each available member's haplotype-tagged reads. See The genome browser (IGV).

How to review

1. Start from the default view of flagged regions.

2. For each region, compare the Haplotype 1 and Haplotype 2 rows with the Combined row. At a normally imprinted region, one haplotype is typically methylated and the other isn't; a region methylated on both copies, or neither, suggests loss of imprinting.

3. Check Mean Coverage and CpGs Observed to make sure the region is well covered.

4. Flag regions showing loss of imprinting or otherwise unexpected methylation, and classify them so they can be included in the report.