In cancer genomics, assumptions have a short shelf life.
Not long ago we published a blog series debunking several common myths about long-read sequencing. At the time, many of those ideas were already starting to show their age as researchers explored specifically what highly accurate long reads could reveal for complex cancer genomes.
Since then, the pace of innovation has only continued to accelerate, and continued advances have reinforced what researchers were already beginning to see. As the body of data continues to grow, new evidence has only pushed those misconceptions further out of date.
Here is an updated look at some persistent misconceptions and what the latest advances now make clear.
Myth: PacBio is more expensive than nanopore sequencing for cancer genomics research applications
Fact: This statement is false.
The cost of highly accurate long-read sequencing is often misunderstood, and the economics only continue to improve.
With the introduction of SPRQ-Nx chemistry, PacBio is pushing long-read sequencing costs to an entirely new level. SPRQ-Nx enables whole genome sequencing at approximately $345 per human genome with 20x coverage while maintaining the high accuracy of HiFi reads. This dramatically lowers the barrier for large-scale cancer genomics projects and makes HiFi sequencing the most affordable long-read whole genome sequencing option available. When combined with the breadth of information captured in a single HiFi dataset including structural variants, repeat expansions, phasing, and methylation signals, the idea that accurate long reads are prohibitively expensive simply no longer holds up.
Myth: Long-read sequencing isn’t compatible with FFPE samples
Fact: This statement is outdated.
Formalin-fixed paraffin-embedded (FFPE) samples have long been considered difficult material for long-read sequencing. Formalin fixation fragments DNA and introduces crosslinking, which historically made it hard to recover molecules long enough for meaningful analysis.
New workflows are changing that reality. Covaris long-fragment extraction methods combined with Kinnex concatenation now allow researchers to generate robust HiFi sequencing libraries from FFPE-derived DNA. Even with fragmented input, these libraries produce high-quality reads with median accuracy reaching Q60 and mean read lengths around 2 to 3 kb. While shorter than typical HiFi reads, these molecules are long enough to enable variant phasing and structural variant detection, two areas where long reads provide clear advantages.
In practice, this means many cancer genes can still be phased effectively. In one evaluation, 68 percent of genes under 50 kb could be phased in a single block using FFPE-derived libraries, compared with 84 percent using high-quality DNA, only a moderate reduction given the fragmented nature of FFPE DNA. The result is a practical path for analyzing archived tumor samples and retrospective cancer cohorts that were previously difficult to study with long-read sequencing.
Myth: Short-read RNA-seq captures all transcriptomic dysregulation in cancer
Fact: This statement is shortsighted.
Recent studies continue to show just how much transcriptomic complexity short-read approaches miss.
Short-read RNA sequencing has been invaluable for gene expression analysis, but fragmented reads often struggle to reconstruct full transcript structures. Long-read RNA sequencing provides a more complete view by capturing full-length transcripts, revealing a layer of transcript diversity that short reads frequently miss.
Multiple studies now illustrate the impact of this difference. In breast cancer, researchers discovered nearly 100,000 previously unannotated isoforms, more than doubling the number detected with short-read methods. In gastric cancer, long-read transcript sequencing revealed alternative promoter usage in ARID1A that produces truncated isoforms that better predict progression-free survival than previously known transcripts, highlighting how isoform-level information can reveal new biomarkers that remain invisible to gene-level analyses. And in colorectal cancer, single-cell long-read RNA sequencing has uncovered tumor-specific isoforms that generate neoantigens with predicted HLA binding, providing potential targets for cancer vaccines.
Together these studies highlight how full-length transcript sequencing can reveal clinically meaningful biology that fragmented reads simply cannot reconstruct.
Myth: HiFi sequencing instruments are too low-throughput for somatic variant detection
Fact: This statement is outdated.
Earlier perceptions about long-read sequencing throughput reflected older platforms and workflows. Today researchers have far more flexibility.
The Revio system is a high-throughput sequencing platform capable of generating whole-genome datasets at the depths required for somatic variant detection, making comprehensive long-read cancer genome analysis far more practical than before. At the same time, the Vega system enables rapid sequencing runs with turnaround times of roughly 24 hours, allowing labs to process samples without waiting to batch large projects while still achieving the coverage required for somatic analysis.
Both platforms can generate whole-genome datasets at depths suitable for cancer studies, enabling accurate detection of somatic variants and complex structural rearrangements.
Myth: HiFi sequencing has complicated bioinformatics and lacks clear protocols
Fact: This statement is inaccurate.
Modern analysis pipelines have made long-read cancer genomics far more accessible. The HiFi Somatic WDL workflow provides an end-to-end pipeline for somatic variant detection from HiFi whole genome data and now supports both matched tumor–normal and tumor-only analyses. Within a single workflow, researchers can identify SNVs, indels, structural variants, copy number alterations, repeat expansions, and native CpG methylation while also calculating tumor mutation burden, microsatellite instability, homologous recombination deficiency, and mutational signatures. Bringing these features together, the pipeline produces a first-pass interactive report alongside standard outputs for deeper analysis.
New tools are also expanding specialized analyses. For example, the recently introduced Owl method enables precise quantification of microsatellite instability directly from long-read genomic data, improving the ability to detect this important cancer biomarker and study its role in tumor evolution and treatment response.
As tools like these continue to emerge, the bioinformatics ecosystem around HiFi sequencing is making it easier than ever for researchers to extract meaningful insights from cancer genomes.
Myth: Long-read sequencing is only a research tool and doesn’t meet the needs of modern clinical research
Fact: This statement is outdated.
Real-world studies are increasingly showing the clinical value of comprehensive long-read genome analysis. At Children’s Mercy Kansas City, clinicians investigating pediatric leukemia cases used HiFi whole-genome sequencing to analyze patients whose cancers remained unexplained after standard testing. Traditional approaches including cytogenetics, FISH, microarray, and short-read sequencing had already been applied, yet roughly 10 percent of cases still lacked a clear genetic explanation.
HiFi sequencing helped close that gap. In this cohort, whole-genome sequencing revealed cryptic structural rearrangements in about one-third of previously unresolved cases, including clinically significant rearrangements involving genes such as KMT2A and ZNF384. These findings highlight genetic drivers that deepen our understanding of pediatric cancer biology and may ultimately help guide clinical care for families facing uncertainty.
In related work presented at ACMG, researchers at Children’s Mercy Kansas City also demonstrated that long-read sequencing can reliably detect clinically relevant somatic structural variants in pediatric cancers, achieving complete concordance with established testing while resolving complex breakpoints. Together, these results illustrate how HiFi sequencing is expanding what researchers can uncover in cancer genomes.
What HiFi sequencing makes possible
Cancer genomics continues to move quickly, and the technologies used to study it are evolving just as fast. Many of the myths that once surrounded long-read sequencing were rooted in earlier technical limitations or incomplete information and are only increasingly outdated. Advances in sample preparation, sequencing throughput, and analysis workflows are expanding what researchers can learn from a single experiment.
To see how HiFi sequencing is helping researchers uncover new insights across cancer genomics, explore our cancer genomics page or experience recent discoveries through Cancer breakthroughs, an interactive HiFi experience.