How does HiFi sequencing work?
HiFi sequencing is a single-molecule, real-time sequencing technology (SMRT) that provides incredible single-molecule read accuracy across long reads of tens of kilobases in length or more. HiFi reads are generated by combining information from multiple observations of a single DNA molecule, resulting in over 99% accuracy of individual HiFi reads.
Developed by PacBio, this process occurs inside small wells on a special microchip called a SMRT Cell, which contains millions of these tiny wells. HiFi sequencing uses fluorescent light signals to identify DNA bases and modified bases (without bisulfite treatment). As a polymerase enzyme adds new nucleotide bases to a newly replicated strand, it emits tiny flashes of light. This technology powers our HiFi long-read sequencing platforms, providing reliable long reads and accurate results.
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What are the advantages of HiFi sequencing?
Long reads
With reads tens of kilobases in length you can readily assemble complete genomes and sequence full-length transcripts. HiFi sequencing provides exceptional read lengths without compromising throughput or accuracy.
Data from an HG002 human library using the SMRTbell express template prep kit 2.0 on a Sequel IIe system (2.0 chemistry, Sequel IIe system software v10, 30-hour movie). Read lengths, reads/data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size.
High accuracy
Sequencing free of systematic error achieves 99.9% read accuracy and >90% of bases Q30+. Explore the benefits of highly accurate long-read sequencing. With exceptional accuracy, low sequencing-context bias, and accurate mapping of reads, HiFi sequencing provides the information needed to confidently call and detect all variants.
Data from a 15 kb size-selected human library using the SMRTbell express template prep kit 2.0 on a Sequel IIe system (2.0 chemistry, Sequel IIe system software v10, 30-hour movie). Read lengths, reads/data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size.
Uniform coverage
No bias based on GC content means you can sequence through regions inaccessible to other technologies. Readily sequence through AT-rich or GC-rich regions, highly repetitive sequences, long homopolymers, and palindromic sequences with HiFi sequencing.
Mean coverage per GC window across a human sample. Data generated with a 20 kb HiFi library on a Sequel II system (2.0 chemistry and Sequel II system). Read lengths, reads/data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size.
Native molecules
Capturing sequence data from native DNA or RNA molecules enables highly accurate long reads with >99.9% single-molecule accuracy. Readily achieve high-quality reads to confidently resolve variants of all types.
Data from a 15 kb size-selected human library using the SMRTbell express template prep kit 2.0 on a Sequel IIe system (2.0 chemistry, Sequel IIe system software v10, 30-hour movie). Read lengths, reads/data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size.
Epigenetics
With no PCR amplification step, base modifications are directly detected during sequencing. Measurement of variation in polymerase kinetics of DNA base incorporation eliminates the need for chemical modification to detect base modifications. This allows you to capture sequence and epigenetic information in a single experiment. See how on-instrument 5-base HiFi sequencing detects methylation without additional library preparations.
Frequently asked questions about how HiFi works
HiFi sequencing offers a streamlined, end-to-end workflow—from sample to analysis.
Start with high-molecular-weight DNA extraction using Nanobind DNA kits, delivering quality DNA from a wide range of sample types in about two hours. DNA is then converted into SMRTbell libraries using manual or automation-friendly prep kits.
Libraries are loaded onto Vega or Revio systems, with simple run setup in SMRT Link software.
After sequencing, advanced algorithms—including Google Health DeepConsensus accelerated by NVIDIA GPUs—process data onboard to generate highly accurate HiFi reads. Outputs are delivered in standardized BAM format, ready for genome assembly, variant detection, phasing, and other downstream analyses.
This unified workflow reduces complexity and accelerates time to insight.
The total time from DNA sample to HiFi reads typically ranges from a few days, depending on the application and system used.
- DNA extraction: ~2 hours using Nanobind kits
- Library preparation: ~1 day
- Sequencing run: Hours to ~1 day, depending on insert size and system (Vega or Revio)
- Data processing: onboard processing is performed automatically, generating highly accurate HiFi reads shortly after the run completes
Because HiFi reads are produced with Q30+ accuracy, downstream analysis often requires fewer correction steps compared to other long-read methods—helping researchers move quickly from raw DNA to actionable biological insight.
Outputs are delivered in standardized BAM format, ready for genome assembly, variant detection, phasing, and other downstream analyses.
HiFi sequencing does not require PCR amplification because it is a single-molecule, real-time (SMRT) sequencing technology. Instead of copying DNA millions of times before sequencing, HiFi reads are generated by observing a single native DNA molecule as a DNA polymerase synthesizes it in real time.
Each DNA fragment is converted into a circular SMRTbell template. During sequencing, the polymerase makes multiple passes around the same molecule. These repeated observations are combined through Circular Consensus Sequencing (CCS) to produce a highly accurate HiFi read (Q30+), without the need for amplification.
Avoiding PCR provides several advantages:
- Reduced amplification bias, including improved representation of GC-rich or complex regions
- More uniform genome coverage
- Preservation of native DNA characteristics
- Direct detection of base modifications
By achieving accuracy through consensus of multiple passes—rather than amplification—HiFi sequencing delivers long, highly accurate reads while maintaining the integrity of the original DNA molecule.
HiFi data combine long read lengths (typically 15–25 kb) with Q30+ accuracy, they can be used directly in many standard bioinformatics workflows:
- De novo genome assembly – Produces highly contiguous assemblies with fewer gaps and more complete resolution of repetitive regions.
- Structural variant (SV) detection – Accurately identifies large insertions, deletions, inversions, and complex rearrangements.
- Small variant calling (SNVs and indels) – High accuracy supports confident detection of single-nucleotide and small insertion/deletion variants.
- Haplotype phasing – Long reads span multiple variants, enabling accurate phasing across extended genomic regions.
- Full-length transcript sequencing (Iso-Seq analysis) – Captures complete transcript isoforms without assembly.
- Epigenetic modification detection – Native DNA sequencing enables direct detection of base modifications.
Because HiFi reads reduce ambiguity in complex genomic regions, they simplify pipelines that traditionally required hybrid sequencing approaches or extensive validation—accelerating time from data generation to biological insight.
Explore popular tools and workflows for HiFi applications
Explore example datasets with the applications and tools
