Q&A: PacBio HiFi genome sequencing to uncover rare disease mysteries

Rebekah Jordan fires some questions with Neil Ward, vice president and general manager for Europe, Middle East, and Africa, PacBio, to learn of the trends of 2023, and how its HiFi genome sequencing technology will be used in collaboration with Boston Children's Hospital to uncover novel mutations in rare paediatric diseases.

Q. What trends do you expect to see in biopharma 2023?

We expect to see significant innovation in 2023 in cancer research. Genomic sequencing plays a central role in cancer – from diagnosis, to allowing us to better understand its complex biology and progression at a molecular level, to pain management and treatment advice. Recent years have seen significant advances in the development of both long- and short-read genomic sequencing. It is these advances that will drive innovation and accelerate the path of research and treatment towards a precision medicine approach.

For example, the latest long-read sequencing machines deliver higher throughput and affordability than has been previously possible. This edges us closer to being able to integrate highly accurate long-read data into cancer research at scale. In short-reads, the increased sensitivity and specificity in today’s sequencing systems will drive breakthroughs in the development of diagnostic tools to improve therapy selection and recurrence monitoring.

Q. What challenges have you seen in AAV sequencing and how has PacBio overcome them?

Adeno-associated viruses (AAV) are typically preferred for delivering DNA to target cells due to their low toxicity, dependence upon other viruses for replication, broad tropism, and the ability to infect both dividing and non-dividing cells. In cancer research, the key area of interest in AAV is how the method can be used to create new cancer vaccines.

One of the key barriers in developing AAV therapies and vaccines is achieving the right level of accuracy and completeness when sequencing genetic material. The ability to see full length of an AAV is important for the discovery of truncated products, while high accuracy is needed to identify undesired mutations. To date, short reads, qPCR, and less accurate long-read systems have not captured this detail, potentially affecting the safety and efficacy of AAV products or vaccines. PacBio’s HiFi system sequences the entire, intact AAV with high accuracy. This allows for detection of mutations and large structural events to aid the success of novel vector discovery, vector design, and manufacturing quality control for AAV projects.

Q. Could you tell us more about PacBio’s HiFi whole genome sequencing and how it works?

PacBio’s HiFi whole genome sequencing is the only sequencing technology that provides accuracy of 99.9% without compromising on read length. Short-read sequencing has many useful applications, but whole genome analysis is needed to advance areas like large-scale cancer research, where insight into complex genomic changes and “dark” regions of the genome is required.

In the past, attempts have been made to synthetically reconstruct longer molecule sequences by pasting together multiple partial short reads. But this impacts accuracy and molecular integrity. HiFi reads overcome this by extracting whole strands of DNA molecules from cells and directly submitting them to sequencing. Samples are not broken and pieced back together, which ensures accuracy is maintained and offers the fastest time to assembly.

Q. Recently, PacBio started a pilot study at Boston’s Children’s Hospital for HiFi in rare paediatric diseases – what outcomes do you hope to achieve from this?

Researchers for the Children’s Rare Disease Cohorts Initiative (CRDC) at Boston Children’s Hospital are using PacBio’s HiFi whole-genome sequencing to investigate genetic and epigenetic variants associated with rare paediatric diseases. Researchers will be using samples from individuals who have already enrolled in multiple rare disease cohorts, but remain their diseases remain unexplained after short-read sequencing efforts.

Our goal is to use PacBio solutions to explain rare disease mysteries where other technologies cannot and support the development of diagnostics for many of these families. The overall aim is to uncover novel mutations, so researchers can start developing targeted therapies and match people with relevant clinical trials.

In previous studies of a similar kind, HiFi sequencing detected more than two-fold structural variants and 200,000 more single-nucleotide variants than traditional short-read sequencing. In some cases, these structural variants contributed to twice the number of new explanations for rare diseases, and we hope to bring this same success to the studies being done at Boston Children's Hospital.

Q. What do you think the future holds for pharmacogenomics (PGx)?

Hundreds of millions of pounds are wasted administering drugs that are not efficacious due to an individual’s genetic makeup. In the future, we expect to see PGx screening projects set up with the aim of reducing this loss, ensuring people can metabolise a drug, and of predicting adverse events.

We also know that pharmacogenomics is very relevant and applicable in the field of oncology. Particularly in pain management and ensuring patients receive adequate pain relief during treatment. But also, through DPYD (dihydropyrimidine dehydrogenase) gene testing, which is used to predict response to fluoropyrimidines therapies commonly used in chemotherapies. The PGx insights that can be gained here can potentially help ensure patients receive dosages tailored to their needs or an alternative therapy. High accuracy in this area of research is therefore vital.