This month’s April 2025 roundup features new research using PacBio HiFi sequencing to investigate unresolved questions in human health, evolution, and agriculture. From rethinking the genetic cause of a rare neurodevelopmental disorder to building some of the most complete ape genome references to date, these studies reflect how researchers are applying long-read data to areas where short reads have left gaps.
Also featured: new benchmarks for small variant detection in clinical samples, deeper isoform analysis in the brain, and early insights into cfDNA modifications that could support future applications in cancer research.
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RID | Plant + animal | Human genome research | Cancer research
RID
In this publication, researchers from Bioscientia and U Cologne conducted a study that’s “findings reversed our previous molecular diagnosis”.
Key highlights:
- Researchers previously reported on a family with severe Joubert syndrome (JBTS) with early-onset severe retinal dystrophy (EOSRD) and polycystic kidney disease (PKD), attributing the condition to a homozygous pathogenic missense variant in the POC1B gene.
- However, when follow-up cases with POC1B variants didn’t align with the same syndromic presentation, the team revisited the index patient using HiFi whole genome sequencing (WGS). This led to the discovery of a homozygous deep-intronic variant in the nearby CEP290 gene—located 1.3 Mb from POC1B—that is predicted to disrupt CEP290 function.
- These new findings strongly suggest that the clinical features, including EOSRD and PKD, are driven by the CEP290 variant. The POC1B variant likely plays only a minor role in non-syndromic retinopathy. As the authors conclude: “Our novel findings in this family no longer justify POC1B as a JBTS gene.”
Conclusion:
HiFi sequencing on the Revio system enables comprehensive phasing and structural characterization of CYP21A2 variants, outperforming short-read sequencing and optical genome mapping. By consolidating multiple tests—including long-range PCR, Sanger sequencing, and MLPA (Multiplex Ligation-dependent Probe Amplification)—into a single, streamlined workflow, “WGS with long reads … allows for extensive haplotype phasing and immediate awareness of an in cis constellation of two pathogenic recessive variants. In view of its superior quality, we expect long-read WGS to replace “conventional” short-read WES and WGS in the near future.”
Plant + animal
Complete sequencing of ape genomes
In this landmark study published in Nature and led by UW and alongside 59 additional institutions, researchers established “a comprehensive baseline for future evolutionary studies of humans and our closest living ape relatives”.
Key highlights:
- Researchers generated haplotype-resolved reference genomes for six ape species using a combination of the T2T workflow and the Verkko assembler. The result: chromosome-level assemblies with exceptional sequence accuracy—fewer than one error per 500,000 base pairs—and the complete telomere-to-telomere sequencing of 215 previously incomplete chromosomes.
- “Each genome assembly was annotated by NCBI [including use of 50 Gb of full-length cDNA generated from each sample with Iso-Seq] and has been adopted as the main reference in RefSeq, replacing the previous short- or long-read- based, less complete versions of the genomes and updating the sex chromosomes with the newly assembled and polished versions.”
- Beyond assembly, the team conducted extensive analyses to refine evolutionary insights. These included revisiting the widely cited ~99% sequence identity between humans and chimpanzees, revealing up to a 15-fold higher difference in certain rapidly evolving genomic regions. Additional analyses covered speciation timing, incomplete lineage sorting, gene and repeat annotations, mobile element insertions, and detailed investigations into immunoglobulin/MHC loci, epigenetic features, structural variation, chromosome rearrangements, segmental duplications, and lineage-specific gene families.
Conclusion:
To date, only long-read sequencing (LRS) has been able to access the 10–15% of highly divergent, previously unreachable regions of ape genomes—regions that are essential to understanding complex traits. By delivering high-quality, haplotype-resolved reference genomes, LRS deepens our understanding of both evolutionary and functional differences across ape species, with meaningful implications for human health. Looking ahead, the newly uncovered genomic regions from this study will continue to require LRS to be fully resolved.
Human genome research
Analytical validation of germline small variant detection using long-read HiFi genome sequencing
In this study, researchers from Stanford find that “HiFi genome sequencing outperformed short-read genome sequencing on overall SNV/indel F1-score accuracy at all paired sequencing depths”.
Key highlights:
- Using HiFi whole genome sequencing (WGS) on both GIAB reference samples and clinical specimens, researchers observed exceptionally high accuracy: “F1-scores for SNVs and indels surpassed 99% at ~15× and ~25×, respectively.”
- Reproducibility was also high across two specimens and three independent sequencing datasets. “Reproducibility assessments (two specimens, three independent sequencing datasets) were >99.8% for SNVs and >98.6% for indels, and average high confidence small variant concordance between paired blood, saliva, and swab specimens were all >99.8%.”
- “Taken together, these data underscore that long-read HiFi genome sequencing detection of SNVs and indels is very accurate and robust, which supports the implementation of this technology for clinical diagnostic testing.”
Conclusion
The findings highlight that “long-read HiFi genome sequencing detection of SNVs and indels is very accurate and robust, which supports the implementation of this technology for clinical diagnostic testing.”
Cancer research
In this study, researchers from Hong Kong found HiFi sequencing to be “a versatile tool expanding the applications of single molecule real-time sequencing in liquid biopsies”.
Key highlights:
- Their updated transformer-based model showed improved detection performance for 5mC, and now includes the ability to identify both 5hmC and 6mA modifications.
- When applied to long cell-free DNA (cfDNA), the model enabled “enhanced detection of patients with hepatocellular carcinoma, with an AUC of 0.97. Moreover, HK model 2-based detection of 6mA enables the detection of jagged ends of cfDNA and the delineation of cellular chromatin structures.”
Conclusion
These findings highlight the growing potential of HiFi sequencing combined with advanced machine learning models to unlock new dimensions of cfDNA analysis, offering powerful tools for cancer detection, epigenetic profiling, and future applications in precision medicine.
Ready to make discoveries of your own?
The April 2025 publications show how HiFi data played a central role in uncovering hidden intronic mutations, building telomere-to-telomere genome references, benchmarking small variant accuracy, and exploring cfDNA methylation patterns – all with the goal of generating clearer insights across research areas.
Whether you’re working in rare disease, neurobiology, evolutionary genomics, or cancer research, these studies reflect how more complete and accurate data can shift perspectives and, sometimes, even rewrite the molecular story.
Stay tuned for the next edition of Powered by PacBio as we continue to highlight how scientists around the world are using HiFi sequencing in their research.
Curious what HiFi could reveal in your work? Let’s get started