PacBio HiFi long-read sequencing technology has made huge strides in helping us understand tumor biology, from the ability to call more variant classes in tumor genomes, to understanding the way these genetic changes are expressed at isoform-level resolution. However, certain questions in cancer and liquid biopsy research may not necessitate long-read sequencing when genomic material in the blood, like circulating tumor DNA (ctDNA), is composed of primarily short fragments.
See how an important new PacBio short-read sequencing chemistry called sequencing by binding (SBB) has the potential to help revolutionize liquid biopsy and cancer diagnostic research by exploring:
- The development of liquid biopsy as a potential method for cancer detection.
- The extraordinary capabilities of SBB sequencing with the Onso system.
- How SBB can dramatically enhance ctDNA detection for a new generation of liquid biopsy assays.
Liquid biopsy: a quick, noninvasive glimpse into the cancer genome?
Liquid biopsy involves sampling bodily fluids like blood to look for biological indicators that can help guide medical care. Such tests have wide applicability in areas as varied as cancer treatment, heart disease, immune disorders and more. Applying liquid biopsy techniques to tackle cancer research has garnered widespread attention because of its potential to noninvasively reveal cancer prognosis, progression, and potential treatment responses. At present, the most prominent cancer indicators (also known as biomarkers) are cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) as well circulating tumor cells (CTCs), metabolites, and proteins. Of these biomarkers, the analysis of ctDNA has been the most active and extensively studied area of research using liquid biopsy.
However, correctly identifying ctDNA in the blood is no easy task. Picking out a cancer variant is even harder than trying to find a needle in a haystack, it is like trying to find a few slightly tarnished needles in an ocean of otherwise identical needles. Herein lies the central challenge for making an effective liquid biopsy assay; and this is precisely why the capabilities of SBB sequencing with the PacBio Onso system holds so much promise for future development in this field.
Setting the stage for a new generation of liquid biopsy research powered by Q40+ accuracy
Since the launch of the first FDA approved assay for oncology in 2016, liquid biopsy research and development has made significant strides. Early tests based on real-time PCR could only determine the presence or absence of tumor variants at a single gene locus and were unable to provide insights into allele frequency. To get around this limitation, subsequent assays integrated dPCR (digital PCR) giving researchers access to the quantitative information they needed. However, this approach still restricted assessments to a few loci at a time. Today, scientists developing liquid biopsy assays can take advantage of targeted NGS sequencing panels to probe hundreds of variants at once and leverage multi-omic techniques to examine tumor associated DNA fragmentation patterns, methylation status, and more. As promising and innovative as such approaches may be, nearly all are based on short-read sequencing technologies whose underlying accuracy has remained largely unchanged for more than a decade, until now.
SBB and the making of liquid biopsy revolution
Unlike traditional sequencing by synthesis (SBS) technologies that have been used for liquid biopsy so far, SBB uses optimized conditions for each phase of the sequencing cycle. By separating the binding and extension steps of the polymerization process specifically, PacBio scientists and engineers were able to create a sequencing chemistry that practically eliminates raw read errors. The resulting dramatic boost in sequencing accuracy has the potential to solve a critical problem for liquid biopsy research and development: How do you tell the signal from the noise, the cancer variant from normal DNA?
The answer: create a better short-read sequencing technology. With SBB and Onso — PacBio has done just that.
Bringing liquid biopsy into the accuracy era
Prior to the release of the Onso system, short-read sequencing had an error rate of approximately 1 in 1,000 bases or Q30 which is insufficient for ultra-precise ctDNA detection in liquid biopsies. As a result, scientists have turned to complex computational error-correction and massive amounts of sequencing to try and circumvent the problem and have been met with only limited success.
SBB sequencing blows this technical logjam completely out of the water by enabling extraordinary Q40+ accuracy. This translates to an error rate of 1 in 10,000 to even 1 in 100,000 bases in certain cases. That is a 10 to 100-fold increase in accuracy over what has come before!
This great leap forward in sequencing precision means that researchers working on liquid biopsy development for cancer applications will be able to take advantage of a considerably higher signal to noise ratio in SBB data to enhance and improve the effectiveness of their assays. The Q40+ accuracy of SBB on the Onso system is the key innovation that can enable users to call extremely rare ctDNA variants more confidently with dramatically fewer confounding errors introduced by the system than other known sequencing technologies.
Testing SBB on a ctDNA standard
For example, in a two-panel comparison using the SeraCare ctDNA reference standard, the new SBB-based Onso system vastly outperformed conventional synthesis-based sequencing with nearly double the sensitivity for rare variant detection even with equivalent sequencing depth. PacBio scientists also found that adding unique molecular identifier (UMI) based deduplication –a type of barcode-based technique for removing duplication errors– increased the sensitivity of SBB sequencing even further. In addition, the Onso system achieved equivalent or better performance compared to older technologies using 4-fold less sequencing. In keeping with the scientific tradition of openness and collaboration, PacBio has made these preliminary SBB ctDNA datasets openly available to anyone wishing to verify these results for themselves.
As any medical researcher reading this article knows, achieving the utmost sensitivity and specificity is crucial for the success of a medical technology like liquid biopsy and the well-being of the people it is meant to serve. And while SBB and the Onso system cannot be used to directly treat or diagnose cancer or any other type of disease, together they represent the critical breakthrough sorely needed by those building the next generation of liquid biopsy assays that might one day be used to do just that.
Tools for disruption and a call to action in the application of genomics for the greater good
Delivering paradigm-shifting short-read sequencing solutions like SBB chemistry on the Onso system to the liquid biopsy space is just the beginning. PacBio strives to create disruptive genomic tools that empower scientists to make discoveries and create innovations that will drive new revolutions in biology. Whether you are an aspiring student, an established scientist, or a curious person working in an allied field, now is the time to take these tools and push the limits of what is possible with genomics. With innovations like SBB, researchers are gaining new platforms for making discoveries so advanced as to be, as writer Arthur C. Clarke once said, “sufficiently indistinguishable from magic.”