Sequencing 101: Comparing long-read sequencing technologies

Last updated: 16 April, 2026

 

In the rapidly advancing field of genomics, choosing the right long-read sequencing technology is key to meeting your experimental goals smoothly and successfully. To make an informed decision, it’s important to understand how different long-read data types, namely HiFi reads and nanopore reads, stack up against each other based on your project’s specific needs—like accuracy, application fit, cost, and resources. Here are a few examples of how these choices play out in real research scenarios:

• Human genomic studies: HiFi sequencing is your best bet if your project needs high accuracy to identify genetic variants and mutations. HiFi sequencing combines long read lengths with exceptional accuracy, even in the challenging “dark” regions of the genome, such as GC-rich or repetitive areas. HiFi sequencing can accurately call and phase both small and large variants, providing crucial haplotype information essential for disease research.
• Rapid pathogen identification: During disease outbreaks, speed is everything. Nanopore sequencers offer a rapid setup and real-time data generation, making it possible to quickly identify pathogens. This capability makes it a valuable tool for scientists and healthcare professionals managing infectious disease outbreaks.
• Resolving complex genomic variation: HiFi sequencing is particularly effective for detecting structural variants, tandem repeats, and other complex regions that are difficult to analyze with short-read or inferred long-read approaches. This is especially valuable in applications such as rare disease research, where accurately resolving complex variation can be critical for identifying potentially causal variants.

This comparison of long-read sequencing technologies highlights key differences between HiFi and nanopore sequencing across accuracy, read length, and application fit.

 

What is long-read sequencing and why choose it over short-read solutions? 

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Long-read sequencing produces genomic data by generating individual reads that are thousands of nucleotides or more in length. These reads typically come from “native” DNA or RNA from a biological sample, preserving any base modifications present. On the other hand, most short-read sequencing technologies use fragments that are 50 to 300 bases long and rely on enzymatic amplification to generate sufficiently large clonal populations, which introduces bias and loses information about base modifications.

In recent years, there have been efforts to extend short-read sequencing by reconstructing longer sequences from shorter fragments. Approaches like Illumina’s TruPath workflow aim to provide long-range genomic information by ¹. While these methods can offer insights in certain human whole genome sequencing applications, they rely on computational reconstruction rather than directly observing long DNA molecules. As a result, they may face challenges in fully resolving complex structural variation, repetitive regions, or complete haplotypes compared to native long-read sequencing approaches.

These approaches can provide additional context compared to standard short reads, but they do not fully replicate the accuracy and completeness of native long-read sequencing.

 

 

Comparing PacBio HiFi and Nanopore Technologies

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How does Nanopore sequencing work? 

Nanopore sequencing technology, popularized by Oxford Nanopore Technologies, involves passing a single strand of DNA or RNA through a protein nanopore embedded in a membrane. A voltage applied across this membrane causes ions to flow through the pore, creating an electric current. The electrical current changes based on the piece of DNA or RNA passing through the tiny pore, with numerous nucleotides affecting the signal at the same time.^2,3,4 To determine the DNA sequence from these signals, scientists match the changes in the current to known patterns of short DNA pieces. Because the current doesn’t show the identity of individual nucleotides, nanopore sequencing can sometimes make mistakes in calling indels within repetitive regions, where it can lose track of the sequence.

Nanopore sequencing can also be used to detect modified bases, since these modifications alter a nucleotide’s ability to influence current flow. This presents both an opportunity and a challenge. On the one hand, it provides an opportunity to detect a flexible set of possible base modifications. On the other hand, modified bases expand the set of possible answers to the question “what sequence explains my current signal?” making basecalling more difficult. Users often need to choose a basecalling model that balances sensitivity to likely modifications with overall accuracy and speed.

While nanopore sequencing offers flexibility and real-time analysis, its lower raw read accuracy and systematic error profile can require higher coverage to achieve comparable results.

 

Figure adapted from Wan et al. (2022)⁵

 

How does HiFi sequencing work? 

To see how HiFi sequencing works in practice and how it enables highly accurate long reads for comprehensive variant detection and multiomic insights, watch the video below.

 

How HiFi sequencing works: From sample to results, see how this approach delivers high accuracy, robust variant detection, and integrated genomic and epigenomic insights in one run.

 

HiFi sequencing is unique because it is very accurate, with a 99.9% accuracy, comparable to other methods like short reads and Sanger sequencing. Developed by PacBio, 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 process occurs inside millions of small wells on a special microchip called a SMRT Cell.

 

 

Each DNA molecule is read multiple times as it is sequenced, allowing a highly accurate consensus to be generated from repeated observations of the same template. This helps reduce random errors and results in HiFi reads with exceptional base-level accuracy. In practice, combining long read lengths with high accuracy allows researchers to confidently detect a full spectrum of genetic variation, including single nucleotide variants, indels, and structural variants, while also preserving long-range haplotype information. These capabilities make HiFi sequencing particularly powerful for real-world applications such as rare disease research, cancer genomics, and de novo genome assembly, where both completeness and precision are essential.

 

Key advantages of HiFi sequencing include:

• High accuracy: HiFi reads typically exceed 99.9% accuracy, enabling confident variant detection across the genome.
• Long read lengths: Reads of 15–20 kb or longer allow researchers to span repetitive and complex regions.
• Comprehensive genomic insight: HiFi sequencing captures both sequence variation and epigenetic information, including 5mC, 5hmC, and 6mA + chromatin accessibility
• Cost efficiency: The Vega benchtop platform makes HiFi sequencing more accessible, priced at USD $169,000 and ~$1,100 per run. The latest SPRQ-Nx sequencing chemistry on Revio enables SMRT Cell multi use, significantly improving cost efficiency and lowering the cost of a 20x human genome to ~$345.

 

How SPRQ-Nx chemistry is improving the cost and scalability of HiFi sequencing

Recent advances in PacBio sequencing chemistry are making HiFi sequencing more affordable than ever before. By enabling multiple uses per SMRT Cell while maintaining output per run, SPRQ-Nx chemistry improves efficiency and lowers cost without adding workflow complexity. Now delivering a 20× human genome at approximately $345, HiFi whole genome sequencing is no longer restricted to small cohorts or premium budgets.

At the same time, these advances are expanding the multiomic capabilities of HiFi sequencing, including support for 5hmC detection for more comprehensive epigenetic profiling. These capabilities complement existing epigenomic approaches like Fiber-seq that enables single-molecule analysis of chromatin structure and genome organization alongside sequence variation.

See how SPRQ-Nx compares in performance and economics in a against leading sequencing technologies including nanopore sequencing and TruPath.

Compare here

 

Choosing the appropriate long-read solution for your project 

 

When choosing a long-read technology platform, consider the following: 

• Accuracy and read length: Different technologies offer varying levels of accuracy and read lengths, which impact the quality of sequencing results. 

• Application suitability: Some platforms are better suited for specific applications, such as detecting structural variants, RNA sequencing, or de novo genome assembly. 

• Cost and resources: Budget constraints and available resources may influence your choice, as some platforms are more cost-effective or resource-intensive than others. 

• Data analysis: The chosen technology influences the bioinformatics tools and pipelines required for data processing and analysis. 

 

Table showing comparison of long read technologies  

 

The long and short of it 

The choice of sequencing technology is an important one, and it depends on the specific needs of your project, such as read length, accuracy, cost, and application. For impactful science, HiFi sequencing is often the best fit due to its high accuracy and comprehensive insights. Beyond technical benefits, PacBio has a global and world-class service and support team that provides personalized assistance throughout the HiFi sequencing process, supporting scientists every step of the way.

 

Ready to get serious about long-read sequencing? 

Compare the cost and performance of HiFi sequencing directly with these competitors in our head-to-head analysis

Learn more about PacBio HiFi sequencing:

Explore other posts in the Sequencing 101 series

Understand the HiFi difference and debunk common sequencing myths

See customer success stories for yourself

See if HiFi sequencing can be applied to your applications

• • Human genomics
  • Microbial genomics
  • Plant and animal genomics
  • Cancer genomics
  • Cell and gene therapy research

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References

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