March 28, 2024  |  Human genetics research

Leading the way into the future: HiFi whole genomes as a replacement for exome and short-read genome sequencing

Abstract image representing the exome

For years, exome sequencing has been a popular method for identifying disease-causing variants, both in clinical and research settings, due to its relative affordability and practicality when compared to whole-genome sequencing. However, because the exome is confined to only protein-coding regions of the genome (about 1.5% to 2%), it is inherently limited.1 That limitation has long been an acceptable trade-off for cost and throughput reasons, but it no longer needs to be.

HiFi whole-genome sequencing on the Revio system not only captures a more complete picture of genetic variation, both in introns and exons, but also delivers the ability to detect the types of variants that short-read technologies have traditionally struggled to detect.2,3 The accuracy of HiFi sequencing could one day replace exomes and short-read whole genomes in the not-too-distant future.

Children’s Mercy Kansas City, one of the USA’s leading pediatric health organizations, and Germany’s Bioscientia, an international clinical laboratory diagnostics company, are leading the way. They have developed methods that use HiFi sequencing for genetic disease research and analysis, resulting in a faster time for results and a more complete view of disease-causing variation compared to short-read whole exomes and whole genomes.

Current limitations of exome sequencing

It’s true that exome sequencing can help end diagnostic odysseys, but even with exome sequencing, only a minority of patients receive a definitive molecular diagnosis.4

Despite its clinical utility, the limitations of exome sequencing are well documented. Because it captures only ~2% of the genome, it excludes many potential disease-causing variants that fall outside exons. Recent research indicates that pathogenic mutations can occur in deep intronic regions, which would be missed by exome sequencing.5

Exome sequencing is limited not only by being confined to coding regions, but also by its lack of coverage uniformity (especially in high-GC areas). Exome sequencing also has limited success in detecting certain types of large variants, such as structural variants.

To address these shortcomings, some laboratories turn to whole-genome sequencing. As we discuss in the following section, short-read whole-genome sequencing is still subject to limitations that miss certain types of disease-causing variation.


A contradiction in terms: how short-read “whole genomes” fail to capture the whole picture and why PacBio long reads are the answer for the future

When exome sequencing fails to uncover a causative variant for a disease phenotype, laboratories may use whole-genome sequencing as a second-line test. However, some have begun to question this approach due to a growing body of literature showing that the clinical utility of short-read genomes and exomes are not significantly different.6,7

Short-read whole genomes still encounter some of the same challenges in detecting large or complex structural variants, as well as variants in highly repetitive or GC-rich regions. Research suggests that the extent and complexity of structural variation has largely been underestimated, until now.7

In the face of these challenges, long-read sequencing offers a better method for capturing genomic complexity, especially for the potential identification of disease-causing structural variation that might be missed with short-read exomes or whole genomes.8 This is especially true in rare disease research, where the wealth of extra genetic context provided by long reads is helping explain cases that were unexplainable with short-read technologies.9 Children’s Mercy and Bioscientia are making this promise a reality by replacing short-read whole-exome approaches with long-read sequencing in their research.

Children’s Mercy and Bioscientia lead the way with PacBio long-read sequencing adoption for a brighter tomorrow

At the forefront of this glimpse into the future, Children’s Mercy and Bioscientia are using the potential of long-read HiFi sequencing to accelerate the time to results by capturing multiple variant types in a single sequencing run, without requiring first-line exomes and second-line whole genomes. At Children’s Mercy, HiFi sequencing has become the new first-line test in their research, replacing other conventional testing modalities.

Dr. Pastinen and the team at Children’s Mercy are using 5-base HiFi sequencing that delivers highly accurate reads with annotation of 5mC methylation at CpG sites and no special library preparation like bisulfite treatment required. This allows them to capture all known mechanisms.

“Results that can take months with multiple legacy tests can now be achieved in two weeks with HiFi sequencing,” says Dr. Pastinen.


Hanno Bolz, MD, Head of Bioscientia’s Human Genetics Division, and the team at Bioscientia have launched a clinical whole-genome sequencing test using HiFi long reads on the Revio platform.

Dr. Bolz notes that long-read sequencing with HiFi technology reads has several advantages over traditional exome or short-read whole genomes, including:

Long-read sequencing can better resolve complex exonic regions or complex variation within an exon.

Long-read sequencing has been able to detect variants in highly repetitive regions, which are difficult to analyze using short-read or Sanger sequencing.

Long-read sequencing also includes phasing information, which can be especially important in rare disease cases.

Dr. Bolz considers long-read sequencing “the way to go,” for many rare disease research studies, and notes that many who administer whole-exome sequencing will move to a long-read test in the future. The capabilities of HiFi sequencing on the Revio system are making that future possible.

Genomic everything, everywhere, all at once

The research teams at Children’s Mercy and Bioscientia demonstrate how the capabilities of HiFi sequencing are evolving the way we think about the utility of the genome. With the throughput of the Revio system and the high accuracy of HiFi long-read sequencing, researchers now have access to far-reaching genomic data much more easily — and with fewer experiments — than traditional short-read sequencing allows.

The potential of HiFi sequencing on the Revio system doesn’t stop there. This powerful technology is also making it possible to characterize the “multiome.” A single sequencing run on the Revio system can generate not just a high-quality whole genome, but multiple different ‘omes all at once.

In a study titled “Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition,” researchers describe the simultaneous generation of four ‘omes — the genome, CpG methylome, chromatin epigenome, and transcriptome — all from a single Revio SMRT Cell sequencing run.

The Revio system allows researchers to assess the same types of variants that can be seen with traditional short-read sequencing (such as SNPs), while adding the ability to access the rest of the genome, methylome, and more —without the need for multiple additional experiments.

short-read WGS vs. long-read WGS comparison table

A growing bioinformatics ecosystem for a whole new genome

Generating data from all those ‘omes at once still requires tools for analyzing it all. PacBio is here to help make it easier for researchers to make impactful discoveries and translate that genomic data into insights.

In addition to the Revio system, PacBio has built comprehensive SNP + SV detection workflows and released new analysis tools, including:

PacBio WGS Variant Pipeline — a best-practice workflow for alignment, variant calling, joint calling and optional annotation.
TRGT — for tandem repeat genotyping and visualization.
Paraphase — a caller for highly homologous genes.
HiFiCNV — a copy number variant caller and depth visualization utility.
PBSV — a suite of tools to call and analyze structural variants in diploid genomes from HiFi reads.
HiPhase — for phasing both small and structural variants from HiFi sequencing data.


How much does a HiFi whole genome cost?

As the teams at Children’s Mercy and Bioscientia have demonstrated, one HiFi sequencing run allows you to consolidate multiple tests into one. Compared to the alternative, which requires running multiple tests, each with their own associated time, material, and labor costs, HiFi sequencing offers many advantages.

Keeping this in mind, for about 1000 USD* per 30x human genome on the Revio system, a single sequencing SMRT Cell can give a valuable, more complete view of variation as a first-line test, without requiring follow-on tests. The Revio system is optimized to produce one 30x whole genome per SMRT Cell with a ~24-hour turnaround time, which means you do not have to wait to “batch” samples to hit that cost target.

Jump into tomorrow and get more for less with HiFi whole genome sequencing

The world of genomics is changing. Long reads, which many regarded as an immature technology, are now being hailed as the Method of the Year. HiFi sequencing is being recognized for population-scale applications, notably for its “widespread value for establishing the most complete and accurate variant calls for All of Us and potentially for many other projects.”

Children’s Mercy and Bioscientia are leading the way, but this is only the beginning. Please connect with us to discuss how your project can benefit from PacBio HiFi sequencing on the Revio system and experience the future of highly accurate whole-genome sequencing, today.



  1. Venter JC, The sequence of the human genome. Science. 2001 Feb 16;291(5507):1304-51. doi: 10.1126/science.1058040. Erratum in: Science 2001 Jun 5;292(5523):1838. PMID: 11181995.
  2. De Coster, W., Weissensteiner, M.H. & Sedlazeck, F.J. Towards population-scale long-read sequencing. Nat Rev Genet 22, 572–587 (2021).
  3. Ebbert, M.T.W., et al. (2019) Systematic analysis of dark and camouflaged genes reveals disease-relevant genes hiding in plain sight. Genome Biol., 20, 97
  4. Marwaha S, Knowles JW, Ashley EA. A guide for the diagnosis of rare and undiagnosed disease: beyond the exome. Genome Med. 2022 Feb 28;14(1):23. doi: 10.1186/s13073-022-01026-w. PMID: 35220969; PMCID: PMC8883622.
  5. Vaz-Drago, R., Custódio, N. & Carmo-Fonseca, M. Deep intronic mutations and human disease. Hum Genet 136, 1093–1111 (2017).
  6. Clark, M.M., Stark, Z., Farnaes, L. et al. Meta-analysis of the diagnostic and clinical utility of genome and exome sequencing and chromosomal microarray in children with suspected genetic diseases. npj Genomic Med 3, 16 (2018).
  7. Kingsmore SF, Cakici JA, Clark MM et al. A Randomized, Controlled Trial of the Analytic and Diagnostic Performance of Singleton and Trio, Rapid Genome and Exome Sequencing in Ill Infants. Am J Hum Genet. 2019 Oct 3;105(4):719-733. doi: 10.1016/j.ajhg.2019.08.009. Epub 2019 Sep 26. PMID: 31564432; PMCID: PMC6817534.
  8. Merker JD, Wenger AM, Sneddon T, et al. Long-read genome sequencing identifies causal structural variation in a Mendelian disease. Genet Med. 2018 Jan;20(1):159-163. doi: 10.1038/gim.2017.86. Epub 2017 Jun 22. PMID: 28640241; PMCID: PMC5741540.
  9. Cohen, Ana S.A. et al. Genomic answers for children: Dynamic analyses of >1000 pediatric rare disease genomes Genetic in Medicine Vol 24 Issue 6, 1336-1348 (2022)

* Study design, sample type, and level of multiplexing may affect the number of SMRT Cells required. Costs may vary by region. Pricing includes library and sequencing reagents run on the Revio system and does not include instrument amortization or other reagents.

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.