This month’s publication highlights show how HiFi sequencing is helping researchers tackle challenges across clinical genomics research. See how HiFi is helping researchers sequence ultra-low input cancer samples, profile methylation more comprehensively than bisulfite sequencing, resolve elusive repeat expansions and discover novel isoforms in cancer immunotherapy research.
Jump to topic:
Low-input sequencing | Epigenetics and methylation | Repeat expansion disorders | Cancer immunotherapy
Low-input sequencing
Whole-genome variant detection in long-read sequencing data from ultra-low input patient samples
In this preprint, researchers from Baylor, Fox Chase PA, Stanford, and PacBio applied an ultralow-input (ULI) protocol to assess genome-wide variation with only 10 ng of input DNA.
Key highlights:
- The ULI protocol outperformed MDA-based amplification when benchmarked against GIAB standards.
- “ULI-HiFi also successfully illuminated several medically important genes that were poorly mapped with short-read DNA sequencing.”
- Applied to samples with hereditary colorectal cancer – including normal tissue, polyp, and adenocarcinoma – the approach revealed progressive tandem repeat expansion in a tumor suppressor gene.
Conclusion:
This work demonstrates how ultralow-input HiFi sequencing expands access to clinically relevant samples, enabling comprehensive variant detection even when DNA is limited. The protocol has since been refined into the Ampli-Fi protocol, which reduces input requirements further to just 1 ng. Combined with flexible library prep on Revio and Vega systems, HiFi sequencing now offers exceptional versatility while maintaining its hallmark accuracy and completeness.
Epigenetics and methylation
In this study, researchers in Thailand compared PacBio HiFi sequencing against whole-genome bisulfite sequencing (WGBS) to evaluate methylation detection in a twin cohort.
Key highlights:
- The authors noted that “Studying DNA methylation using monozygotic twins is uniquely advantageous, as they serve as well-matched controls for nearly all genetic variations and a wide range of environmental factors.”
- Finding “HiFi WGS identified ~5.6 million more CpG sites…than WGBS”, particularly in repetitive elements and regions of low WGBS coverage.
- In CpG sites, “HiFi WGS exhibits an increase of approximately 3.2 million mCscompared to WGBS.”
- They found that coverage patterns differed markedly: “PacBio HiFi showsa unimodal and symmetric pattern peaking at 28–30 × , indicating relatively uniform coverage. In contrast, both WGBS datasets display right-skewed distributions, with the majority of CpGs covered at low depth (4–10×) and relatively few achieving higher coverage. Over 90% of CpGs in the PacBio HiFi dataset have ≥10 × coverage, compared to approximately 65% in the WGBS dataset.”
- HiFi enabled de novo DNA methylation analysis, reporting CpG sites beyond reference sequences.
- Overall, the authors conclude: “Our findings support the reliability of HiFi WGS for methylation detection and highlight its advantages in regions that are challenging for bisulfite-based methods.”
Conclusion:
Because HiFi sequencing interrogates the entire genome, it also delivers a more complete view of the epigenome, capturing millions of additional CpG sites and enabling de novo methylation analysis without chemical conversions or extra workflows. With 5-base sequencing available directly on Revio and Vega, researchers can integrate methylation profiling into every run as part of standard HiFi data.
Repeat expansion disorders
In this study, researchers from Vanderbilt used HiFi whole-genome sequencing to “establish a long-sought molecular [answer] in a family affected by Familial Adult Myoclonic Epilepsy type 3 (FAME3).
Key highlights:
- A 61-year-old subject with refractory seizures had undergone multiple rounds of nondiagnostic exome and genome testing.
- HiFi WGS “identified a pathogenic MARCHF6 intronic expansion” (one allele with 15 TTTTA repeats and a second allele with a compound expansion of 661 TTTTA and 12 TTTCA repeats).
- “Three affected relatives shared similarly expanded alleles, but with increasing repeat size in the latter generations”.
- Authors note that the “disease seems to arise when TTTCA repeats occur in tandem with TTTTA motifs, suggesting a composite structure”. “This highlights the diagnostic importance of assessing both repeat length and motif composition when evaluating suspected repeat expansion disorders.”
- And finding “using TRGT-instability revealed repeat mosaicism in all affected individuals, reflected by variability in motif counts across individual sequencing reads.”
Conclusion:
HiFi sequencing revealed the complex repeat expansion structure driving disease that were missed by conventional testing. The study highlights the value of long-read sequencing for repeat expansion disorders, as well as the ability of TRGT-instability to detect mosaicism at single-molecule resolution. On Revio and Vega, HiFi WGS combined with TRGT tools provides researchers and clinicians with a powerful framework for understanding difficult repeat-associated conditions.
Cancer immunotherapy
Intron retention regulates STAT2 function and predicts immunotherapy response in lung cancer
In this study, researchers from JAX, UConn Health, Immunoledge NJ, Hartford Cancer Institute, and Genentech applied the PacBio Iso-Seq method to explore how alternative splicing influences immune responses in lung adenocarcinoma.
Key highlights:
- Authors highlight that “lung cancer is a leading cause of death globally” – “While immune checkpoint inhibitors have improved disease prognosis, many patients do not benefit, and existing biomarkers fail to predict responses adequately”.
- They also note that “Splicing is often altered in cancer to favor oncogenic and immune-evasive isoforms, but the mechanisms underlying these changes in LUAD are poorly understood”. And finding that “Long- read sequencing … enables deeper functional analyses than would be possible utilizing conventional short-read RNA-sequencing (short- read sequencing) technology”.
- Using the Iso-Seq method, researchers mapped the landscape of alternative RNA splicing in human primary lung adenocarcinomas. We identified over 180,000 full-length mRNA isoforms, more than half of which were noveland many of which occurred in immune-related genes”.
- Discovering “retained introns in the STAT2 gene that produce altered protein isoforms that regulate immune signalingand interferon responses.
- “Furthermore, STAT2 intron retention levels predicted patient responses to checkpoint inhibitors.”
Conclusion:
The Iso-seq method doubled the number of isoforms detected compared to short-read RNA-seq, revealing splicing events with direct relevance to cancer immunotherapy. The authors note that long-read RNA sequencing “may hold the key to breakthroughs that lead to the next wave of therapeutic advances.” With current workflows using Kinnex kits and the Revio system, such insights can now be achieved at scale and reduced cost, requiring only half a SMRT Cell to generate equivalent data compared to 12 SMRT Cells in the study.
Ready to bring accuracy to your research?
August’s studies show how HiFi sequencing makes it possible to sequence ultra-low inputs, capture methylation in full, resolve repeat expansions, and reveal novel isoforms, all with the accuracy and versatility needed to drive research into precision medicine ahead.
Curious to see what HiFi could do in your lab?