In collaboration with Pacific Biosciences (PacBio), the USF College of Public Health’s Genomics Program  and Sequencing Core recently hosted a Genomics Symposium entitled “Long Read Single Molecule Sequencing: A New Era in Genomics.”

Pacific Biosciences, commonly known as PacBio, is a biotechnology company that develops and manufactures cutting-edge systems for gene sequencing, a process that allows scientists to determine what genetic information is carried in a particular segment of DNA.

The PacBio Discoveries Roadshow provided the audience with a valuable opportunity to explore tools that have the potential to fuel the next breakthroughs in understanding genetic diseases, identifying novel genetic variations and exploring the complexities of the human genome, revealing functional elements and accelerating disease research. These tools can also offer advancements in areas such as microbiome research, environmental genomics and understanding symbiotic relationships.

This year’s roadshow includes stops in North America, Europe, South America and Asia with over 40 events on the schedule from March to May. Scientists were again able to come together, listen to talks from local researchers using PacBio sequencing technologies and hear about the new PacBio products and applications.

On Tuesday, May 16, PacBio’s Discoveries Roadshow brought them to Tampa, where over 70 people came together in the USF Research Park Building to hear about the latest updates in PacBio sequencing technologies. Emily Reynolds, territory account manager at PacBio, stated that USF Research Park in Tampa was an ideal location to host this research conference event.

Talks included representatives from PacBio discussing innovations in applications and instrumentation, as well as three scientific talks from local southeast researchers.

PacBio’s revolutionary sequencing technologies combine the completeness of long reads with the accuracy of short reads, offering the most comprehensive view of genomes, transcriptomes (transcriptomes represent the complete set of RNA molecules transcribed from the genome) and epigenomes (the collection of modifications and factors that modify gene expression within a genome).

With HiFi reads, which provide an accuracy of 99.9 percent and up to 25 kb length sequencing, PacBio is the only sequencing technology that allows for the accurate detection of various types of variants, from single nucleotides to structural variants, which can uncover complex genomic structures, identify structural variations and provide a more comprehensive understanding of genomes.

Most single-cell experiments are done with short reads, which only capture the ends of molecules due to fragmentation. Sequencing fragments limit expression information to the gene level, missing important isoform diversity that could be important for disease or biological function. 

Dr. John Adams, director of the USF Genomics Program and co-director of the Center for Global Health and Infectious Diseases Research, presented his research on the functional analysis of gametocyte development in human malaria parasites (gametocytes differentiate into male and female gametes, crucial for malaria transmission).

His talk focused on the utilization of long-read single-cell RNA sequencing to characterize the full-length Plasmodium transcripts from the malaria parasite. This approach, empowered by PacBio MAS-Seq (Multiplexed Arrays Sequencing method) for single cell isoform sequencing along with 10x single-cell barcode and UMI information, enables the identification of stage-specific differentially expressed isoforms (different forms of the same proteins).

Dr. Chengqi Wang, a research assistant professor in the USF Genomics Program and a computational expert in genomics data analysis and the single-cell isoform analysis field, highlighted the significance of mRNA isoforms (mRNA are molecules that carry the genetic information cells need to make proteins).

“Many genes of the mutant parasite can generate multiple isoforms, and mRNA isoforms can be considered as variations of a recipe to prepare different proteins,” Wang stated.

Dr. Michael Kladde, professor of biochemistry and molecular biology at the University of Florida, presented a novel, cost-effective and multiplexed method for achieving high levels of on-target sequencing, allowing for the detection of genetic and epigenetic heterogeneity.

His talk, titled “Precision, flap endonuclease-mediated targeted profiling of genetic and epigenetic heterogeneity on single DNA molecules,” emphasized the utilization of PacBio HiFi long reads at high coverages. This methodology has been instrumental in revealing epigenetic mechanistic insights in cancer, sepsis and other disease conditions. Epigenetic mechanisms can alter the way a gene is expressed.

The keynote speaker, Dr. Jeremy Schmutz, faculty investigator at the Hudson Alpha Institute for Biotechnology AL, showcased the remarkable world of HiFi genomics in their research over the past three years. They have successfully applied PacBio HiFi sequencing to address challenging issues in genome sequencing, including human de novo disease identification (a human de novo disease is a disease not present in either parent but present in their child) and sequencing complex plant genomes. Dr. Schmutz presented early data results from the newest PacBio sequencing platform, Revio.

The Revio long-read sequencing system holds great potential for enabling large-scale research projects with enhanced efficiency. Consequently, researchers will be able to undertake ambitious endeavors more swiftly and cost effectively, while attaining a more comprehensive and precise understanding of structural variations, epigenetic profiles and single nucleotide variants.

Dr. Min Zhang, the USF Genomics Program sequencing core director and one of the organizers of the event, emphasized the capabilities of the PacBio Revio long-read sequencing system and the MAS-Seq for single-cell isoform sequencing.

“The PacBio HiFi read technology overcomes the limitations of short-read sequencing, allowing scientists to delve deeper into unraveling complex biological systems by employing long-read single-cell sequencing,” Zhang said. Researchers can now gain a more comprehensive understanding of genetic diversity, specifically in terms of cell type-specific isoform diversity and the role of isoforms in diseases and biological processes such as cancer or Alzheimer’s.

Moreover, PacBio’s tools provide invaluable assistance in deciphering disease mechanisms, identifying disease-associated variants and uncovering novel drug targets. These advancements have the potential to significantly improve diagnostics, therapies and personalized medicine approaches.

Story by Donna Campisano and Dr. Min Zhang, USF College of Public Health, Genomics Program