The ability to detect DNA methylation with PacBio sequencing may be old news, but the scientific discoveries it enables are anything but.
Since the inception of PacBio long-read sequencing, the detection of methylated bases has been an integral part of the sequencing run, first described over 15 years ago1. Since then, hundreds of peer-reviewed publications in many areas of genomics have described the merit of obtaining contemporaneous epigenetic information during PacBio sequencing, including Entering the era of bacterial epigenomics with single molecule real time DNA sequencing (2013), Widespread adenine N6-methylation of active genes in fungi (2017), Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation (2017), and Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort (2023), to name just a few.
In recent months, additional outstanding preprints and papers have posted which continue to highlight HiFi accurate sequencing and methylation calling in biological samples and their value to genomic research. These include the very first human panepigenome, providing “a foundation for panepigenomic studies across tissues, populations and disease contexts” and revealing how much genetic and epigenetic information is missed when relying on a reference genome.
For a closer look at how HiFi sequencing characterizes methylation and other variation that short reads can miss, explore our new guide and its published examples.
Building the first draft human panepigenome
In Human panepigenome represents epigenomic diversity from researchers of the Human Pangenome Reference Consortium (HPRC), the authors “leverage 440 haplotype-resolved PacBio HiFi methylomes … of 220 individuals … together with near-T2T assemblies and HPRC2 graph coordinates, to construct a first draft of the human DNA methylation panepigenome … spanning 26 globally distributed populations”. This work revealed “12.8 million CpGs absent from GRCh38,” none of which are visible in GRCh38 reference-based short-read bisulfite sequencing by definition.
It also revealed an average of 1.2 million variant-associated CpGs (var-CpGs) per methylome”, again most of which are missed by bisulfite sequence due to a reliance on the reference. Notably, “Var-CpGs captured population-associated epigenomic variation beyond that represented by shared reference CpGs, and SVs could create or remove entire CpG islands.” Also, “promoter var-CpG methylation was associated with transcript expression”, and “CpG-altering variants were also enriched among molecular QTLs in HPRC2 and across GTEx tissues, and among clinically and pharmacogenomically annotated loci”.
Highlighting the importance of combining highly accurate sequencing with methylation calling, the authors conclude that “these results establish a haplotype-resolved framework in which human epigenomic diversity reflects not only variation in methylation level but also genetic variation in presence or absence of CpG substrates”.
HiFi methylation and episignature analysis in rare disease research
The power of accurate, genome-wide methylation calling and its complementarity to genetic information in the context of rare disease and clinical research is further underscored by Genome-wide methylation detection and episignature analysis using PacBio long-read sequencing from researchers at Radboud University Netherlands and Toulouse Hospital France.
Assessing “the ability of PacBio HiFi LRS to robustly identify genome-wide methylation for medical purposes”, they find that “PacBio methylation calls on the HG002 cell line were highly comparable to short-read protocols, providing 5% additional calls, predominantly in regions that are difficult to assess using short-read technologies.” Further, benchmarking 10 trios using 25 known parentally imprinted regions, “correct haplotype-resolved methylation patterns were found for 24 out of 25 (96%) imprinted regions and all of the methylated alleles showed the expected parental origin. The authors conclude that “PacBio LRS can reliably detect specific medically relevant methylation changes as well as genome-wide episignatures in rare disease patients”, and “offers advantages such as phasing information and additional methylation calls in complex genomic regions”.
As in the first paper, this publication also highlights the detection of new CpG sites created by genomic variants. PacBio methylation analysis can be performed in a de novo detection mode which reports all CpG sites found in the consensus builds from reads, not just the CpG sites present in the reference genome. The paper reports over 1.3 million such sites (~4.5% of total), many of which overlap CpG islands and other functional elements. The authors conclude that “PacBio LRS enables the detection of new genomic CpG sites, which might be of particular interest for genomic variants occurring in regulatory genomic regions such as CpG islands or enhancer sequences, where they might modulate the overall epigenetic regional profile”.
Detecting imprinting disorders and episignatures
Additional publications describing the detection of imprinting disorders and epigenetic signatures (patterns of DNA methylation that distinguish individuals with a particular condition from controls) with HiFi sequencing include Sensitivity of HiFi long-read genome sequencing for difficult-to-detect pathogenic variants when applied to real-world clinical laboratory samples (GeneDx & collaborators; Prader-Willi & Angelman syndromes), Clinical Long-Read Sequencing Test for Genetic Disease Diagnosis (Children’s Mercy Kansas City; GNAS methylation), and Yield of Long-Read Genome Sequencing for Rare Disease Diagnosis in Short-Read Genome Negative Cases (Ambry, UC Irvine, GREGoR consortium; Chung-Jansen syndrome).
The analysis of epigenetic signatures is also getting easier. Just last month, the company EpiSign announced support of HiFi data through their EpiSign METRIC 5-base analysis framework for the “automated analysis across more than 300 episignature disorders”. Soon after, the company Geneyx announced incorporation of Episign’s software into their tertiary analysis and interpretation pipeline.
Expanding epigenetic detection from a single HiFi run
We are proud to support our users in their groundbreaking epigenetic studies, and we continue to expand the toolkit of epigenetic detection capabilities. Recently, we added the simultaneous detection of 5-hydroxymethylcytosine – important, for example, in cancers and neurodegenerative disorders – to the output of HiFi sequencing, meaning that Revio and Vega now generate a 7-base output (A, C, G, T, 5mC, 5hmC, and 6mA) directly from the sequencer, and without any offline analysis required. Through the combination of highly accurate HiFi sequencing and methylation detection, we cannot wait to see what epigenetic insights the scientific community will uncover next.
1. Flusberg, B. A., Webster, D. R., Lee, J. H., Travers, K. J., Olivares, E. C., Clark, T. A., Korlach, J. & Turner, S. W. (2010). Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature Methods, 7(6), 461–465. https://doi.org/10.1038/nmeth.1459