PacBio’s Sequel system is based on the same SMRT technology behind the RS II, with a number of improvements. Although we’ve gone over SMRT sequencing chemistry and yield in depth in another blog post, here’s an overview: Single Molecule Real Time (SMRT) sequencing relies on a couple of key innovations. The first of these is a novel approach to phospho-labeling nucleotides. PacBio label their nucleotides on the terminal phosphate that is cleaved during the synthesis reaction. What remains is a completely “natural” DNA double helix. In order to observe this reaction, and actually, perform the base-calling, the polymerase is confined to a nanophotonic chamber - dubbed the Zero-Mode Waveguide (ZMW). This 70nM chamber can observe and quantify the excitation of the phosphate during incorporation. The sequence can consequently be read due to each of the four nucleotides having a distinct fluorescent label.


[1]


The major advantage of SMRT sequencing over other technologies is the long read-length. Other major benefits of the platform, such as improved de novo assembly and detection of structural variation are derived from this advantage. With more and more evidence pointing to the importance of structural variation between different genomes, PacBio’s long reads put it in a unique market position.[2] Other advantages include the ability to faithfully read methylation for epigenetic studies[3], and full-length mRNA for transcriptome studies.[4]

Additionally, certain shorter fragments of RNA, such as ribosomal RNA, can take advantage of PacBio’s SMRTbell adapters. These create an almost circular fragment which can then be read multiple times in the same ZMW. These “circular consensus” (CCS) reads can lead to very accurate base calling, albeit limited in the range of applications.[5]



The Sequel platform is essentially a refinement of the RS II that allows PacBio to scale its SMRT technology. With 1 million ZMW per chip, PacBio claims the platform will offer about 7 times as many reads per SMRT cell as compared to the RS II, with approximately half the up-front cost.[6]

As a result, projects using the platform will enjoy shorter timelines and reduced reagent overheads. With these improvements in mind, the Sequel system has about ⅓ the physical footprint of the RS II. Despite these improvements, limitations inherent in SMRT technology still exist. Amplicon sequencing on the sequel system, for example, is still less accurate than what Illumina can provide - especially for longer amplicons.[7]

Moreover, reagent costs for SMRT sequencing are still higher than Illumina if compared on a cost-per-base basis.[8]

PacBio has an excellent overall platform and chemistry. If they can bring down reagent costs and improve accuracy across all applications, the third iteration of their flagship product will be very competitive in any lab, and indispensable in some.

 




[1] Hashmi, Uzair, et al. "Plant exomics: concepts, applications, and methodologies in crop improvement." Plant signaling & behavior 10.1 (2015): e976152.

[2] Wenger, Aaron, et al. "Structural variant detection with low-coverage PacBio sequencing." Nature 517.7536: 608-611.

[3] Davis, Brigid M., Michael C. Chao, and Matthew K. Waldor. "Entering the era of bacterial epigenomics with single molecule real-time DNA sequencing." Current opinion in microbiology 16.2 (2013): 192-198.

[4]Ashby, Meredith, et al. "Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System." (2017): 2442-2442.

[5]https://www.pacb.com/wp-content/uploads/Vierra-G10K-2017-From-RNA-to-Full-Length-Transcripts-1.pdf

[6]https://www.pacb.com/press_releases/pacific-biosciences-launches-new-sequencing-platform-based-on-its-smrt-technology/

[7]Tedersoo, Leho, Ave Tooming‐Klunderud, and Sten Anslan. "PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives." New Phytologist 217.3 (2018): 1370-1385.

[8]Rhoads, Anthony, and Kin Fai Au. "PacBio sequencing and its applications." Genomics, proteomics & Bioinformatics 13.5 (2015): 278-289.

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