The truth about sequencing accuracy and how PacBio leads the way

 

HiFi sequencing enables next-level accuracy in DNA sequencing. But did you know that level of accuracy extends throughout the PacBio sequencing portfolio—with both short and long reads that raise the bar? Novel sequencing by binding (SBB) chemistry brings remarkable accuracy and low error rates to short-read sequencing too—upending the limitations of yesterday’s technology.

PacBio prioritizes exceptional accuracy across its sequencing menu, so that no matter your research focus, there’s a technology that’s right for you. Let’s explore a few real-world examples that illustrate how PacBio is bringing the “era of accuracy” to a variety of research applications that impact human health and disease.

 

PacBio sequencing accuracy in cancer research

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A coalition of 20 researchers, led by the Genome in a Bottle consortium hosted by the National Institute of Standards and Technology, recently shared a preprint that describes a “first-of-its-kind” public dataset consisting of sequencing data from tumor and matched normal samples. These data will be used to develop tumor-normal benchmarks for somatic variant detection.

Titled “Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair for somatic benchmarks,” the preprint contains detailed data from 13 sequencing technologies, including PacBio. By including a wide array of sequencing providers and detailed quality metrics on each, this study also serves as an evaluation of the accuracy of each of those methods for cancer research applications.

The authors performed whole-genome sequencing using five short-read technologies. A comparison of QC metrics (Table 3) demonstrates that the PacBio Onso system had the lowest mismatch rate of all short-read technologies.

 

The authors also compare three long-read sequencing technologies and show that PacBio HiFi long reads were cleaner and more accurate than the competition, with more bases mapped at higher accuracy than other technology.

This Genome in a Bottle study is among the latest to demonstrate the utility of PacBio sequencing in cancer research, but it is by no means the only one. For more about the accuracy and applications of PacBio sequencing in cancer research, see:

• Why TGen sees big developments coming for cancer liquid biopsy research thanks to ultra-accurate PacBio short reads
• Debunking common myths about HiFi long reads in cancer genomics
• How highly accurate PacBio short reads enable detection of microsatellite instability

 

PacBio sequencing accuracy in neuroscience

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It’s already been proven that long-read sequencing provides more accurate and comprehensive variant detection compared to legacy SBS short reads. How that improvement affects the rate of diagnostic yield, however, has yet to be characterized.

In a recent analysis of a neurodegenerative disease cohort, researchers selected 96 probands with rare diseases that were suspected to be genetic, but came up negative with short-read sequencing. They then re-sequenced these 96 probands using PacBio HiFi long-read sequencing and found new clinically relevant or potentially relevant findings in 16 of them (16.7%), nine of which contained pathogenic or likely pathogenic variants. Of these cases, seven of them (44%) included variants that were only detected by long-read sequencing and completely missed by legacy SBS short-read methods.

The authors conclude that long-read sequencing, conducted on both the PacBio Revio and Sequel IIe systems, “allows for substantial additional [diagnostic] yield” beyond legacy SBS short-read genome sequencing.

 

“Long-read genome sequencing represents the next phase of […] acceleration by facilitating a substantial increase in variant comprehensiveness and accuracy.”

-Hiatt et al. 2024¹

 

For more on the applications of PacBio HiFi sequencing in neuroscience research, see:

• Accuracy of HiFi sequencing supports innovative therapies for Parkinson’s disease
• PureTarget and HiFi sequencing offer new hope for neurodegenerative disease
• How HiFi sequencing is helping researchers tackle ALS challenges

 

PacBio sequencing accuracy in microbiology

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One of the challenges with current NGS technologies is characterizing cellular heterogeneity, including accurate measurement of minor populations due to “the intrinsic error rates, particularly in ‘sequencing by synthesis’ (SBS) methods.”²

In a recently published paper, researchers compared the ability of Illumina SBS vs. PacBio SBB to identify and quantify rare drug-resistant subpopulations in Mycobacterium tuberculosis samples.

In short, the authors found that SBB offers a significant improvement over SBS and rivals error-corrected SBS for accurate variant detection and quantification. SBB detected mutations from 0.01–0.001% frequency at 100,000x depth or >0.1% frequency at 20,000x depth with no error correction methods, a 10x improvement compared to SBS and competitive with error-corrected SBS data. SBB showed an 8.5X reduction in observed errors compared to SBS alone. PacBio SBB sequencing data also showed lower empirical error rates and false positive calls.

The authors propose that SBB offers a powerful alternative over Illumina SBS for detecting rare mutations. Why bother with complex and time-intensive error correction, when you could achieve the same accuracy the first time with SBB?

 

“With lower sequencing error rates within SBB sequencing, [SBB] looks promising for both targeted and unbiased whole genome sequencing, leading to the identification of minor (< 1%) subpopulations without the need for error correction methods.”

-Allender et al. 2024²

 

Reduced error rates, a simpler sequencing process, and highly accurate variant detection for rare variants—these features of SBB make it a critical tool for early detection of heteroresistance, which is essential for tackling drug-resistant tuberculosis. When applied more broadly, SBB offers advantages for other applications that rely on rare variants, such as liquid biopsies or infectious disease surveillance.

For more on the applications of PacBio sequencing in microbiology, read about how scientists are using SBB to understand antimicrobial resistance and protect public health.

 

Sequencing’s era of accuracy is now

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As demonstrated by these examples and more, scientists need no longer be limited by outdated standards of accuracy. Ready to bring the next-level accuracy of PacBio sequencing to your research? Speak to a PacBio scientist about getting started with the Onso short-read system or the Revio long-read system.

Connect with a PacBio scientist

 

References