Epigenetics and DNA modifications with 5-base HiFi sequencing
Epigenetics is about understanding how gene activity changes without altering the DNA sequence itself. Powered by advanced methylation algorithms, PacBio 5-base sequencing simultaneously detects DNA sequences and multiple DNA modifications from native DNA in a single assay. This integrated approach unites long-read phasing and multiomic analysis for a faster, more complete view of biology. Combine this approach with Fiber-seq to map single chromatin fibers and uncover footprints, nucleosomes, variants, and methylation—all in one streamlined assay.
Epigenetics on PacBio at a glance
Video
See the power of HiFi Epigenetics
In this overview video, explore how PacBio highly accurate long reads enable direct detection of base modifications, delivering comprehensive insights into methylation patterns alongside genetic variation. Learn how a single HiFi workflow can illuminate gene regulation, cell differentiation, and disease mechanisms, without the need for additional assays.
Go beyond 5-base sequencing by detecting a broader range of DNA modifications
HiFi long-read sequencing captures DNA methylation by measuring subtle differences in polymerase kinetics during sequencing. As new machine learning models expand to detect additional base modifications (e.g., 5hmC), the power of HiFi sequencing continues to grow—making HiFi sequencing a technology that shapes and evolves with the epigenetics field.
With HiFi, you can:
- Directly link genetic variation with changes in methylation
- Access challenging regions of the genome that defy short reads
- See allele-specific differences in methylation patterns with haplotype phasing
- Detect parental imprinting
“And now, with the introduction of Fiber-seq to characterize nucleosome positioning, the ability to tackle these applications with Vega is a game changer, especially for some of the bigger questions we haven’t yet been able to address –– like how does neuronal development work? How is the immune system triggered in response to certain stimuli? How is regeneration controlled? These are all things we can explore with Vega.”
Andor Kiss from Miami University in Oxford, Ohio
Single chromatin fiber sequencing for high-resolution views of the epigenome
Fiber-seq maps chromatin features at single-molecule resolution on every HiFi read—capturing genetic variation, chromatin accessibility, nucleosome positioning, CpG methylation, and transcription factor binding in one unified assay. This comprehensive, multiomic view reveals haplotype-specific gene regulation and chromatin dynamics that would otherwise require multiple short-read assays.
Key capabilities:
- Single-molecule chromatin accessibility
- Near base-pair nucleosome resolution
- CpG methylation detection
- Transcription factor (TF) footprinting
- Haplotype phasing for chromatin accessibility
- Direct link between genetic variation and gene expression
Epigenetics in action
pb-CpG-tools
The pb-CpG-tools collection provides tools for secondary analysis of CpG methylation data from PacBio HiFi reads. Starting from a pileup of HiFi reads with methylation tags, the tools calculate the percent of reads methylated at every CpG site in the genome.
DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools
Here, we introduce fibertools, a state-of-the-art toolkit that features a semisupervised convolutional neural network for fast and accurate identification of m6A-marked bases using Pacific Biosciences single-molecule long-read sequencing.
Synchonized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition
Resolving the molecular basis of a Mendelian condition remains challenging owing to the diverse mechanisms by which genetic variants cause disease. This article highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.
Breakthrough discoveries in genomics, transcriptomics and epigenomics with HiFi long-read sequencing
This workshop delves into the forefront of genomics research! Discover how innovative researchers are leveraging the extraordinary accuracy and comprehensive coverage of HiFi sequencing to drive groundbreaking insights into the intricate world of genetic variation, RNA splicing, and epigenetic gene regulation.
Explore
Did you know we have over 10,000 articles, reports, papers, and videos related to epigenetic research?
Epigenetic sequencing workflow at a glance
Standard library prep
- No bisulfite or other enzymatic treatment
Fiber-seq library prep
- CUTANA Fiber-seq from EpiCypher
- PacBio standard library prep
Standard library prep
- Simultaneous detection of base sequence (A, C, G, and T) and epigenetic modifications – no additional sequencing experiments need
Analysis
- On-instrument barcoding and methylation calling
- View methylation calls in IGV
- Pb-CpG-tools for methylation pileups
- Fibertools for Fiber-seq data analysis and inferring regulatory elements
Common questions about PacBio epigenetic sequencing
- In eukaryotes, PacBio 5-base sequencing calls 5mC in CpG context directly and 6mA for the Fiber-seq assay.
- In microbes, PacBio can detect 4mC, 5mC, and 6mA using trained kinetic models.
- Ongoing model development aims to expand detection capabilities across more modification types. 5hmC detection will be available with SPRQ-Nx chemistry in 2026.
- HiFi sequencing simultaneously detects base sequences and epigenetic modifications in a single experiment.
- Fiber-seq (6mA) requires an upfront treatment of isolated nuclei with the non-specific adenine methyltransferase Hia5 followed by standard PacBio library preparation.
- HiFi sequencing reveals chromatin accessibility through Fiber-seq, a single chromatin fiber sequencing method. Fiber-seq uses a non-sequence-specific adenine methyltransferase (Hia5) to label accessible regions within intact chromatin.
- The 6mA labels are then detected during standard sequencing allowing for accessible open chromatin regions, nucleosome positions, and TF occupancy to be map across the genome.
- Methylation signatures are called on-instrument during primary data analysis and come included in every sequencing run.
- Alternatively, methylation calling can be performed off-instrument by saving the kinetic data and using the Jasmine software tool
- pb-CpG-tools can be used to generate site methylation probabilities from mapped HiFi reads.
- Fibertools and the FIRE pipeline can be used to interact with Fiber-seq data and infer regulatory elements (accessible regions) and nucleosome occupancy.
FEATURED LONG-READ SEQUENCING SYSTEMS
5-base genome sequencing is now possible. With PacBio long-read sequencers you can gain immediate access to the epigenome with no special workflow or data processing steps required.