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Established in 2004, Pacific Biosciences (PacBio) set out to create innovative tools that would elevate our understanding of genomics. Over the years, they have grown into global leaders in the field, known for their revolutionary HiFi sequencing—a high-accuracy, long-read sequencing solution.
Their journey began with the PacBio RS, their first commercial release, which employed their Single Molecule, Real-Time (SMRT) sequencing technology. This marked the beginning of a series of advances, from the RS II to the Sequel series, each iteration taking strides toward enhanced throughput, read length, and accuracy.
Although an acquisition attempt by Illumina in 2019 fell through1, PacBio has since emerged stronger, significantly improving their instruments and making history as the first company to provide both long- and short-read sequencers. The addition of short-read technology came after the acquisition of Omniome2, accompanied by significant process upgrades. As Mark Van Oene, Chief Operating Officer of PacBio, proudly noted, “I fundamentally believe that we have differentiated technologies for both accurate long reads and accurate short reads. Whether it's DNA or RNA, we're coming up with the technology that allows you to see more.”
According to Van Oene, shifts in the world of genomics are evident in recent papers from the Telomere-to-Telomere (T2T) consortium3,4 and pre-prints from the All of Us Research Program5. He believes these projects highlight the need for a blend of short- and long-read sequencing, emphasizing the value of examining the full genome and understanding the relevance of structural variations, phasing, and epigenetics. In this rapidly evolving sequencing landscape, PacBio continues to provide innovative solutions that enable all types of genomics research.
Available sequencing technologies
PacBio continues to offer a diverse portfolio of cutting-edge sequencing technologies tailored to various research needs. Their current offerings include the Sequel systems, which remain an integral part of their product lineup for tackling complex genomic projects. The newly launched Revio further extends PacBio's long-read sequencing expertise, providing advanced features and performance. Additionally, PacBio has announced the imminent release of their short-read sequencer named Onso, setting new standards in sequencing accuracy and demonstrating PacBio's commitment to innovation and meeting the evolving demands of the genomics research community.
HiFi and SBB
HiFi sequencing, developed and utilized by PacBio, is their advanced long-read sequencing technology that combines the benefits of long-read lengths and high accuracy. This is achieved through the use of their Single Molecule, Real-Time (SMRT) sequencing technology.
This process begins by adding hairpin adapters to double-stranded DNA, producing a circularized SMRTbell library. The library is then loaded onto a SMRT Cell containing nanoscale observation chambers called Zero Mode Waveguides (ZMWs). Within the ZMWs, DNA molecules in the library are drawn to the bottom, where a polymerase enzyme incorporates fluorescently labeled nucleotides in real time. The emitted fluorescence, recorded by a camera, captures the nucleotides' fluorescence and the time intervals between their incorporations, known as the interpulse duration (IPD). Modifications on the DNA strand, such as 6-methyl adenosine, can be detected by delays in IPD compared to non-modified DNA. The library's circular structure enables multiple passes, which generate redundant sequencing reads of the same molecule. These redundant reads are then collapsed into a single consensus sequence, or HiFi read, which significantly improves the accuracy of the sequence data.
PacBio's short-read sequencer, Onso, employs Sequencing By Binding (SBB) as its sequencing technique. The process begins by loading sequencing-ready libraries onto the instrument, which are then loaded on the flow cell. Each sequencing cycle begins with a 3' reversible blocked nucleotide. Fluorescently labeled nucleotides are then introduced to the flow cell, allowing the appropriate base to bind while unbound nucleotides are washed away, reducing the background signal. The 3' end of the nucleotide is activated by removing the reversible terminator. Next, native, unlabeled, reversibly blocked nucleotides are flowed over the flow cell, and the matching base can incorporate into the growing strand. This iterative process is repeated for each newly sequenced base, enabling accurate and efficient sequencing on the Onso sequencers.
Common applications
PacBio’s long-read sequencing instruments, such as Revio and Sequel systems, are uniquely equipped to sequence extensive genomic stretches. This feature is beneficial for various applications, including haplotype phasing, de novo genome assembly, and identifying structural variations. Additionally, these instruments can directly detect methylation, making them ideal for epigenetic studies.
Their capability extends to sequencing full-length transcripts and discovering isoforms. By contrast, short-read sequencing is only able to sequence partial transcripts with one read. Building in this HiFi capability, methods like Multiplexed Arrays Sequencing (MAS-Seq), which concatenates cDNA molecules into longer fragments, significantly enhancing throughput by increasing the total number of full-length transcripts read with one HiFi read. MAS-Seq is also being broadened to include bulk RNA and 16S rRNA sequencing.
PacBio's advancements in long-read technology, especially with the Revio system, also make them adaptable to applications traditionally reserved for short-read technologies. Van Oene underscores that Revio's utility extends beyond being a reference genome technology—it has evolved into a resequencing tool as well. “We talk a lot about it in the context of genomics and genomes, but as a targeted tool, there are so many carrier genes and dark regions we can target. It's not just a whole genome sequencing tool. Revio now enables you to do targeted long reads in a very different way, and on a different scale than you could with Sequel,” added Van Oene. This transformation provides a new approach to targeted long reads.
The incorporation of short-read technology ensures the versatility of PacBio’s sequencers across almost any sequencing application, and the high accuracy of Onso makes it particularly suitable for applications like liquid biopsy that require high specificity and sensitivity. Van Oene explained, “Long reads don't address everything perfectly, and liquid biopsy is one of those really quick emerging areas that we wanted to make sure that we also had a technology to address it.”
Advances and the future
The most significant advancement for PacBio has been the announcement of Onso, their short-read sequencing technology. "We knew we needed a short-read technology, so we started to look around at all the different emerging technologies and look for the chemistry that can be most accurate," explained Van Oene. Currently, Onso boasts an unrivaled accuracy level (Q40), setting it apart in the short-read sequencing technologies. Van Oene again noted that this high accuracy was identified as a critical differentiator to achieving the required sensitivity for liquid biopsy applications.
PacBio has also invested considerable efforts to make Onso user-friendly and ready to use. "Over the last year and a half spent developing that platform, we've been able to improve read lengths, improve densities, and start making this a really meaningful mid-throughput platform to take it to market with that Q40 accuracy," Van Oene stated. He further explained that the device was shifted from single- to paired-end reads, improvements were made to cluster generation, and the user interface was streamlined. “It's a product that the team's going to be really proud of,” he added.
The other big change for PacBio was the release of Revio. “Revio was a massive leap in technological improvement,” said Van Oene. This instrument has 15 times more output than the Sequel systems, which he explained are great sequencing technologies that lacked in throughput. “Being able to do science at scale, that’s driving the interest in Revio.” In addition to the increased throughput, users can expect lower sequencing costs, faster run times, and enhanced computing power that includes Google Health DeepConsensus onboard.
Experiencing a strong demand for Revio sequencers, PacBio is projected to reach double the sequencing capacity of the Sequel IIe install base in just a few short months. Their global showcase, the Discoveries Roadshow 2023, has been an effective marketing tool, highlighting their innovative technologies and their diverse applications. Adding to their self-promotion, the company also benefits from a stream of high-quality research studies featuring their technologies in topics such as pangenomes6, de novo assemblies7, structural variation8, and isoform detection9.
Van Oene explained this growing interest succinctly, "People want to resolve more biology. They want to understand more about the biology of diseases and these tools are starting to unlock that information." Undeniably, PacBio's sequencers are paving the way for a better understanding of complex biological systems, leaving us optimistic about their future contributions to science.
References:
- Federal Trade Commission. FTC Challenges Illumina's Proposed Acquisition of PacBio. https://www.ftc.gov/news-events/pres...isition-pacbio. Published December 17, 2019. Accessed July 3, 2023.
- PacBio. Pacific Biosciences Closes Acquisition of Omniome and Establishes San Diego Presence. https://www.pacb.com/press_releases/...iego-presence/. Published September 20, 2021. Accessed July 3, 2023.
- Nurk S, Koren S, Rhie A, et al. The complete sequence of a human genome. Science. 2022;376(6588):44-53. doi:https://doi.org/10.1126/science.abj6987
- Rhie A, Nurk S, Cechová M, et al. The complete sequence of a human Y chromosome. bioRxiv. Published online December 1, 2022. doi:https://doi.org/10.1101/2022.12.01.518724
- Mahmoud M, Huang Y, Garimella K, et al. Utility of long-read sequencing for All of Us. bioRxiv. Published online January 24, 2023. doi:https://doi.org/10.1101/2023.01.23.525236
- Liao WW, Asri M, Ebler J, et al. A draft human pangenome reference. Nature. 2023;617(7960):312-324. doi:https://doi.org/10.1038/s41586-023-05896-x
- Manuel, JG, Heins, HB, Crocker, S et al. High Coverage Highly Accurate Long-Read Sequencing of a Mouse Neuronal Cell Line Using the PacBio Revio Sequencer." bioRxiv. Published online June 7, 2023. doi:https://doi.org/10.1101/2023.06.06.543940
- Ebert P, Audano PA, Zhu Q, et al. Haplotype-resolved diverse human genomes and integrated analysis of structural variation. Science. 2021;372(6537). doi:https://doi.org/10.1126/science.abf7117
- Al’Khafaji, AM, Smith, JT, Garimella, KV et al. High-throughput RNA isoform sequencing using programmed cDNA concatenation. Nature Biotechnology. 2023. doi:https://doi.org/10.1038/s41587-023-01815-7