Back in April we introduced significant software enhancements to simultaneously provide the base sequence and 5-methylcytosine DNA methylation status by default, making the Sequel II/IIe systems the world’s first (and only) on-instrument 5-base sequencers. The capability allows scientists to gain access to the epigenome with zero additional cost, effort, and complexity. In a new preprint by researchers from Children’s Mercy Kansas City (CMKC) and collaborators, entitled Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort, the immense value of this new feature is powerfully demonstrated. Below is a short summary of the study and its findings:
- The researchers performed 5-base HiFi sequencing of 276 samples from 152 families, and found excellent concordance on the same samples for genomic regions for which short-read whole-genome bisulfite sequencing (srWGBS) data were available.
- They identified over 25,000 rare hypermethylation events that occur in two or fewer individuals. 81% of these were allele-specific, i.e., the methylation outlier occurs in only one allele, and predicted to cause loss of regulatory element activity. This leverages the strength of long & accurate HiFi reads to phase the two haplotypes. The phasing ability resulted in a dramatic increase in the discovery of these events: on average 117 per sample with PacBio, only 8 with srWGBS!
- In terms of the underlying genetics, many hypermethylation events are explained by linked rare SNVs or SVs (i.e., genetic cause of epigenetic difference): SNVs that affect nearby methylation typically are located within 1 kb, SVs typically within 10 kb.
- Linking methylation to disease, on average 30-40 events overlapped rare disease genes per patient, providing indications for variation prioritization. And in one case, methylation outlier analysis identified a previously overlooked causative repeat expansion in a gene causing global developmental delay in the individual.
The work underscores the new opportunities in the field of epigenetics that are empowered by 5-base HiFi sequencing. Their possible connections to clinical research were also described eloquently in an interview by Dr. Tomi Pastinen (CMKC and senior author of the preprint), noting that “this is the first step for linking epigenetic sequencing and DNA sequencing in a high-resolution view of function and genetic variation. This has potential in rare and complex diseases, and perhaps even more with the latter because we know that 90% of susceptibility to common disease is encoded in the regulatory DNA rather than the coding DNA.” Dr. Pastinen further observed that our understanding of the epigenome has been changing in recent years. Whereas most scientists thought epigenetics were “environmental perturbations of the genome for a long time”, this new work shows that “more than half of the epigenetic variation we see across individuals is encoded in the genome in the DNA bases themselves.”, thereby highlighting the powerful impact of 5-base HiFi sequencing to uniquely provide this connection directly in the same sequencing run.
Please connect with us, we look forward to discussing with you how 5-base HiFi sequencing can accelerate your research.