HiFi sequencing with the all new PacBio Revio: Part 1

Revolutionizing personalized medicine – The power of long-read sequencing

The era of personalized medicine

Personalized medicine encompasses an emerging medical approach that adapts healthcare decisions, treatments, and preventive measures to suit the specific characteristics of each individual patient. A crucial aspect of delivering personalized care involves utilizing a patient’s genetic profile as a valuable resource to inform healthcare decisions. This transformative shift in medicine has been made possible by the development of Next-Generation Sequencing (NGS), which has significantly enhanced the speed, accuracy, and affordability of sequencing human genomes and transcriptomes.

However, for personalized medicine to become a widespread reality, it is crucial to undertake large-scale genomic projects involving diverse populations. These serve as critical foundations for understanding and advancing the discovery of pathogenic variants and opening up new avenues for treatments and diagnostics, particularly for rare diseases. Currently, many projects like these still rely on short-read sequencing (SRS) techniques due to their high accuracy, but their limits are increasingly apparent, especially when sequencing complex and highly repetitive regions within a genome [1].

This has led to massive advancements in long-read sequencing (LRS), a technique that generates reads spanning thousands of base pairs, offering enhanced detail and nuance. In this blog, we discuss the necessity of LRS for personalized medicine and how its increased usage could transform healthcare.

Find out how our LRS services can support your research applications

Understanding our genomes

Numerous large-scale projects, such as the Cancer Genome Atlas [2], ENCODE [3], and the 100,000 Genomes Project [4], have undertaken the monumental task of comprehensively mapping the inherent genetic variation within the human genome. Endeavors such as these have been critical to advancements in our understanding of human genetics and have paved the way for improved disease treatments.

The 100,000 Genomes Project, initiated in 2013, aimed to sequence the complete genomes of 100,000 National Health Service (NHS) patients and their families. In 2021, data from this study demonstrated that Whole Genome Sequencing (WGS) led to new diagnoses for 25% of the participants [5]. Remarkably, 14% of these new diagnoses were identified in regions of the genome that would have been missed by other conventional methods, including non-whole genomic tests [5]. This highlights the critical importance of WGS in both the treatment and understanding of diseases.

These initiatives play a vital role in treating rare diseases and cancer, where comprehensively accounting for all genetic variation, from single base changes to large structural variants, is paramount. However, portions of the genome continue to elude us due to the limits of SRS.

Revealing hidden changes

For years, SRS has played a crucial role in improving diagnostic rates for patients with rare diseases. It has been widely adopted as a front-line test in many suspected rare disease cases worldwide. However, despite these achievements, there is still a diagnostic gap that SRS alone cannot bridge. This is where LRS comes into play, enabling us to finally understand, diagnose, and more effectively treat the rare diseases that impact millions of people worldwide.

The utility of LRS was exemplified in the 2018 case of a patient named ‘Ramon’, who faced the challenge of recurrent noncancerous tumors appearing throughout his body [6]. Despite the use of SRS, no variants were identified that could explain his clinical symptoms [6].

However, when LRS was employed, the results revealed a significant discovery—a heterozygous 2,184 bp deletion that overlapped with the first coding exon of the PRKAR1A gene [6]. This gene is associated with autosomal dominant Carney complex, a rare condition characterized by an increased risk of various tumor types, particularly in the heart and hormone-producing glands. Since it is challenging to diagnose, fewer than 750 individuals with this condition have been identified since 1985 [7].

In another notable case in 2021, two monozygotic twins showed symptoms of intellectual disability, yet no pathogenic variants were detected through exome sequencing [8]. LRS was employed to further investigate the underlying genetic causes of their condition. LRS helped to identify a large 12-kilobase (kb) inversion, directly impacting two genes, CPNE9 and BRPF1, which were found to contribute to their intellectual disability [8].

These examples serve as compelling evidence of the capability of LRS to detect complex genomic rearrangements, leading to more precise diagnoses, personalized treatments, and the development of effective disease prevention strategies.

Advantages of long-read sequencing with the PacBio Revio system

The use of LRS in clinical genetics is on the rise, presenting new opportunities for genomic analysis. However, LRS does come with certain limitations, such as lower accuracy and increased cost compared to SRS. To address these challenges, Pacific Biosciences developed HiFi sequencing, which allows for continuous reads spanning 10-25,000 bp while maintaining an impressive accuracy of 99.9%, on par with short reads and Sanger sequencing [9].

One of the latest initiatives in the field of genomics is the ‘All of Us’ program, which aims to sequence the genomes of more than one million Americans representing diverse ethnic backgrounds. The primary objective of this initiative is to enhance personalized medicine.

In a recent analysis that compared the performance of traditional short-read sequencing with long-read sequencing for use in the ‘All of Us’ program, HiFi sequencing emerged as the superior method for accuracy, delivering precise results for both small and large genetic variants [10]. This finding highlights the potential of HiFi sequencing to revolutionize genomic research and contribute to the advancement of personalized medicine.

The PacBio Revio system harnesses the power of HiFi chemistry, enabling the generation of highly accurate native long reads with uniform coverage. The Revio system offers up to a 15-fold increase in throughput compared to existing PacBio systems and can produce a complete, phased genome, at similar price range compared to SRS, making it particularly well-suited for large-scale clinical applications.

Taking genomics beyond the genome

HiFi reads can also provide full length transcript sequencing (Iso-Seq), which grants researchers a comprehensive understanding into disease mechanisms through the detection of gene fusion, and the discovery of novel isoforms. This method can reveal more than traditional DNA sequencing as a gene can be sequenced in multiple forms, including the production of non-coding RNAs.

By employing the Iso-Seq method, researchers have successfully characterized fusion transcripts, copy-number amplifications, and structural variants in SK-BR-3 breast cancer cells [11], revealing previously unknown genetic alterations.  Through the combination of long-read genome and transcriptome sequencing, they gained a more in-depth insight into how structural variants disrupt the genome, shedding new light on the complex mechanisms involved in cancer genome evolution.

By combining high-fidelity sequencing, high throughput, and low error rates, the PacBio Revio system empowers researchers to detect all types of genetic variations, including large structural variants, with remarkable precision. This cutting-edge technology can accelerate genomic research, and ultimately will lead to patients benefiting worldwide.

Explore clinical applications of the PacBio Revio system with Eremid

Large-scale genomic projects will continue to lay the foundation for personalized medicine, transforming healthcare as we know it. With the immense advantages bestowed by LRS, these initiatives will pave the way for increased discovery of pathogenic variants and open new possibilities in diagnosis, treatment, and prevention for rare disorders.

At Eremid, our CLIA-certified laboratory is dedicated to providing accurate and reliable clinical genomics services. Our PacBio Revio system  is the latest generation of LRS systems employing PacBio’s HiFi sequencing technology. With its exceptional capabilities combined with our in-house expertise, we’re poised to help our customers make a profound impact on the future of genomics research and personalized medicine.

Contact us to find out how we can best support your clinical needs!

References

1. Treangen, T.J., Salzberg, S.L., 2012. Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet 13, 36–46. https://doi.org/10.1038/nrg3117
2. The Cancer Genome Atlas Program (TCGA) – NCI [WWW Document], 2022. URL https://www.cancer.gov/ccg/research/genome-sequencing/tcga (accessed 24th of May 2023).
3. The Encyclopedia of DNA Elements (ENCODE) [WWW Document], 2022. URL https://www.genome.gov/Funded-Programs-Projects/ENCODE-Project-ENCyclopedia-Of-DNA-Elements (accessed 24th of May 2023).
4. 100,000 Genomes Project [WWW Document]. Genomics England. URL https://www.genomicsengland.co.uk/initiatives/100000-genomes-project (accessed 24^th of May 2023).
5. 100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care — Preliminary Report, 2021. New England Journal of Medicine 385, 1868–1880. https://doi.org/10.1056/NEJMoa2035790
6. Merker, J.D., Wenger, A.M., Sneddon, T., Grove, M., Zappala, Z., et al., 2018. Long-read genome sequencing identifies causal structural variation in a Mendelian disease. Genetics in Medicine 20, 159–163. https://doi.org/10.1038/gim.2017.86
7. Vindhyal, M.R., Elshimy, G., Elhomsy, G., 2023. Carney Complex, in: StatPearls. StatPearls Publishing, Treasure Island (FL).
8. Mizuguchi, T., Okamoto, N., Yanagihara, K., Miyatake, S., Uchiyama, Y., et al., 2021. Pathogenic 12-kb copy-neutral inversion in syndromic intellectual disability identified by high-fidelity long-read sequencing. Genomics 113, 1044–1053. https://doi.org/10.1016/j.ygeno.2020.10.038
9. Wenger, A.M., Peluso, P., Rowell, W.J., Chang, P.-C., Hall, R.J., et al., 2019. Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nat Biotechnol 37, 1155–1162. https://doi.org/10.1038/s41587-019-0217-9
10. Mahmoud, M., Huang, Y., Garimella, K., Audano, P.A., Wan, W., et al., 2023. Utility of long-read sequencing for All of Us. https://doi.org/10.1101/2023.01.23.525236
11. Nattestad, M., Goodwin, S., Ng, K., Baslan, T., Sedlazeck, F.J., et al., Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line. Genome Res. 28, 1126–1135. https://doi.org/10.1101/gr.231100.117