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Epigenetics with long-read sequencing

The unique chemistry of long-read HiFi sequencing technology enables researchers to directly reveal the epigenetic landscape of samples. On-instrument 5-base HiFi sequencing detects 5mC methylation in standard sequencing runs without any changes to library prep or sequencing workflows required.

HiFi sequencing provides accurate DNA base calls and simultaneous 5mC detection in CpG context without any additional library preparation. This feature enables the resolution of methylation profiles with phased haplotyping. Human genome researchers may also use this capability to interrogate imprinting disorders and methylation abnormalities associated with tandem repeats.

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Human and Eukaryote Epigenetics

Use 5-base HIFI sequencing to study epigenetics of humans and other eukaryotes. Measure 5mC methylation at CpG sites directly from the sequencing instrument without the need for bisulfite treatment or other special library preparation methods.

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Microbial epigenetics

Build a more comprehensive picture of how microbes use epigenetic modification for immune evasion by characterizing DNA modifications and methyltransferase recognition motifs for 4mC and 6mA.

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Application Brief


Genome-wide detection and phasing of genetic and epigenetic variants from a single library prep. HiFi sequencing of a single sample detects methylation patterns across the genome, such as hypomethylation at transcription start sites. Sequencing multiple samples identifies differential methylation.

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HiFi sequencing provides accurate genetic and epigenetic information from a single sequencing library.

Methylation microarrays Short-read sequencing Nanopore sequencing HiFi 5-base sequencing
Haplotype phasing


5mC in CpG contexts
Requires special library preparation
Requires special data processing. Conflated with basecalling
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PacBio HiFi sequencing simultaneously calls the four DNA bases and 5mC from untreated genomic DNA. Achieve genome-wide detection and phasing of genetic and epigenetic variants from a single, standard HiFi library prep with long and accurate reads.

With the power of long-read sequencing, you can achieve:

Epigenetics in every run — no bisulfite treatment required

Unlike methods that require chemical conversion of DNA, HiFi sequencing detects modifications in native DNA through impacts on the kinetics of base incorporation.

High accuracy of sequence and methylation

Methylation detection with HiFi sequencing is highly concordant to bisulfite sequencing.

Access the full genome

Access difficult regions of the genome like repeats and centromeres that are beyond the reach of short-read sequencing.


Identify allele-specific methylation, whether due to parental imprinting, genetic variation, or repeat expansions.

In the example shown below, a repeat expansion on one allele can be seen to impact adjacent methylation status.

HiFi sequencing phases and identifies hypermethylation of the region adjacent to a mosaic 5 kb DMPK expansion in a sample with myotonic dystrophy (Children’s Mercy Kansas City). Magenta indicates methylation, while blue indicates unmethylated bases.

Figure 7. HiFi sequencing phases and identifies hypermethylation of the region adjacent to a mosaic 5 kb DMPK expansion in a sample with myotonic dystrophy (Children’s Mercy Kansas City). Magenta indicates methylation, while blue indicates unmethylated bases.

“SMRT sequencing is opening up new diagnostic avenues, such as the ability to determine tandem repeat lengths, interruptions, and even epigenetics in a single test at base pair resolution.”1

– Ardui, et al., 20181

Epigenetic analysis in action

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Direct detection of DNA methylation

See how scientists use PacBio sequencing to detect methylation with basepair resolution.

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Genome-wide detection of cytosine methylation

Read how researchers use the kinetics in HiFi reads to determine methylation status at CpG sites.

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DNA 5mC detection and methylation phasing

Read how circular consensus sequencing enables genome-wide detection of cytosine methylation by single molecule real-time sequencing.

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Did you know we have over 10,000 articles, reports, papers, and videos related to epigenetic research?

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HiFi sequencing provides two channels of information: fluorescence and kinetics. Utilizing both enables highly accurate reads (fluorescence) plus methylation status (kinetics) from a single library.

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How 5-base sequencing works showing nucleotide incorporation kinetics measured in real time and directly detecting dna modifications during sequencing - PacBio

HiFi sequencing observes a polymerase incorporating fluorescently labeled nucleotides complementary to a native DNA strand. The label identifies the base (A, C, G, T). Epigenetic modifications like 5mC impact polymerase kinetics — how fast bases are incorporated. No special library prep is required.

  • A convolutional neural network model processes polymerase kinetics to determine the methylation status of each CpG site in a HiFi read.
  • The model runs directly on the Sequel IIe system and is also available in SMRT Link.
  • Methylation status is output using the BAM standard MM and ML tags.

Image of Kinetics 5 base showing kinetics in 16bp window around CpG site in read and convolutional neural network and probability of methylation - PacBio


5mC at CpG sites is the predominant epigenetic mark in vertebrates like humans. It is also useful — though not comprehensive — in other eukaryotic species such as plants, which employ 5mC methylation in CpG contexts alongside other motifs. For microbes, which employ a wider variety of modifications, alternative analysis tools are recommended.

Methylation Species HiFi 5-base sequencing
5mC at CpG sites Human + other vertebrates
5mC at various motifs Other eukaryotes, including plants Useful though partial view
4mC and 6mA Microbes Enabled through SMRT Link microbial genome analysis

What 5-Base Sequencing Reveals

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HiFi methylation patterns - PacBio

Regional methylation patterns

Methylation levels vary across the genome in many species. In vertebrates like human most CpG sites are methylated. Active gene transcription start sites are often hypomethylated.

In this example genomic region, 5-base HiFi sequencing of the human HG002 sample shows overall hypermethylation (red) with hypomethylation (blue) specifically at transcription start sites.

Phased genetics and epigenetics

HiFi sequencing enables simultaneous phasing of reads into maternal and paternal haplotypes and detection of methylation. This reveals allele-specific methylation patterns, which can be due to genetic variation (where epigenetic status is affected by a difference in sequence) or parental imprinting (where epigenetic status is affected by whether a chromosome was inherited from the mother or father).

In this example, the HG002/3/4 trio from Genome in a Bottle, HiFi reads show the expected maternal imprinting at the gene PEG3. HiFi sequencing allows phasing of the haplotypes per sample, the trio identifies which allele is transmitted from which parent, and 5-base sequencing shows allele-specific methylation.

imprinting methylation from son, father and mother - PacBio

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Atypical methylation patterns of expanded FMR1 allele comparing HiFi reads vs. position of FMR1 repeat - PacBio

HiFi sequencing phases and identifies hypermethylation of expanded FMR1 repeats in NA07537.

Methylation and disease

Atypical methylation patterns contribute to rare diseases like Prader-Willi syndrome and are important factors in pathogenic repeat expansion, such as the CGG expansion at the FMR1 locus that cause Fragile X syndrome. With high accuracy, long reads, and methylation detection, HiFi sequencing is ideal for characterizing these repeat expansions.

Epigenetic sequencing workflow at a glance

Standard Library Prep

  • No bisulfite or other enzymatic treatment

Library prep kits

Standard sequencing run

  • Simultaneously detect accurate base sequence and accurate epigenetic modifications

SMRT sequencing

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Simple Analysis

  • Detect microbial base modifications and motifs with the microbial genome analysis application in SMRT Link
  • Call 5mC at CpG sites directly from the sequencing instrument or in SMRT Link
  • Visualize 5mC annotation directly in IGV

Common questions about PacBio epigenetic sequencing

A wide range of epigenetic marks have distinct signatures in HiFi sequencing. SMRT Link currently provides models for 4mC, 5mC, and 6mA for microbial genomes. For human and other eukaryotic genome, 5-base HiFi sequencing provides calls for 5mC at CpG sites.

No. Currently 5-base HiFi sequencing only calls 5mC without distinguishing from 5hmC.
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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.

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