This month’s publications show just how versatile HiFi sequencing has become. In September, studies ranged from building the most comprehensive map yet of human genomic imprinting, to evaluating clinical whole-genome sequencing as a potential first-line diagnostic tool, to uncovering hidden hemoglobin variants in large population cohorts and tracking RNA isoforms that could open the door to new cancer immunotherapies.
Across these areas HiFi sequencing is revealing layers of biology that were previously invisible to standard methods. Keep reading for a closer look at this month’s featured research:
Jump to topic:
Methylation | RID | Population carrier screening | Cancer immunotherapy
Expanded map of genomic imprinting reveals insight into human disease
In this preprint, researchers from Children’s Mercy Kansas City (CMKC) find that “HiFi genome sequencing for single-molecular profiling of 5-mC, together with pedigree-based phasing in early developmental tissue, provides critical insight into previously uncharted loci in the human genome.
Key highlights:
The authors first highlight the limitations of traditional methods: In short-read bisulfite sequencing, “only ~7% of reads are informative for allelic effects”. In contrast, “HiFi-GS technology generates 5-mC profiles genome-wide from standard sequencing libraries of long reads (∼16 kb) increasing phasing efficiently over tenfold as compared to WGBS. This improved haploid resolution is “critical for POE [parent-of-origin effect] discovery.” To address this, the team used HiFi WGS on “75 samples from 25 trios” and developed “a 10-fold enhanced map of human imprinting during development”, identifying “52,786 autosomal CpGs in 5852 bins showing POE of CpG methylation (POE-me) of which 60% have previously not been linked to or suggestive of imprinting.” Building on these results, they integrated “summary statistics from population GWAS” and found “enrichment of common (birthweight) and rare (congenital anomalies) disease loci in newly identified imprinting regions.” The study also leveraged pedigree-based rare disease cohorts to show “preponderance of paternal inheritance of pathogenic variants mapping to autosomal dominant OMIM genes with a maternal POE-bin,” identifying two genes (BNC2, DNMT1) as novel candidate imprinting disorder loci. The authors find that “Our enhanced human map of POE of 5-mC significantly extends the current “imprintome” and uncovers previously underappreciated genes and variants that appear crucial for human development and disease.”
Conclusion: Alle-specific methylation is often underappreciated but plays a crucial role in understanding what drives development and disease. Short reads miss most of this signal (up to 60%), proving methylation isn’t just extra data, it’s essential for discovery. With HiFi, you get it automatically with every genome.
Clinical long-read sequencing test for genetic disease diagnosis
In another study by CMKC, the authors, when assessing the potential implementation of long-read sequencing as a first-line clinical test, report “the comparative diagnostic yield and turnaround time and evaluate the ability to consolidate standard-of-care (SOC) approaches using a single clinical LRS test”
Key highlights:
The team performed a clinical study of HiFi WGS on 235 pediatric patients (ages 0–18), comparing results to an age- and phenotype-matched control cohort of 513 patients. Compared to the control group, HiFi sequencing demonstrated a significantly higher diagnostic finding (37% vs. 27%) and a faster turnaround time (27 days vs. 62 days). Even among negative cases, LRS reduced the average time to report from 91 to 29 days. These improvements reflect “the integrated capability of LRS, which included aberrant methylation, rare expansion disorders, phasing of single-nucleotide variation in a singleton, and detection or refinement of SVs.” The authors note, “these benefits of clinical LRS are likely only the beginnings of what may be uncovered through expanded testing and further understanding of noncoding regions.” In final, the authors state that when comparing long-read sequencing case results to controls: “Clinical LRS offers a single, comprehensive genomic assay for diagnosing genetic disease. The advantage of LRS was explained by its expanded variant detection.”
Conclusion: This landmark study demonstrates how HiFi sequencing can transform pediatric disease discovery by delivering 10% higher success over all prior testing methods, helping to provide families with results in <1 month vs. 3, and reducing the need for multiple stressful and costly rounds of testing.
In this study, researchers out of China report that “SMRT sequencing enables efficient and reliable analysis of common and complex or rare hemoglobin variants”:
Autosomal recessive hemoglobinopathies are “characterized by a high degree of clinical and genetic heterogeneity (over 1800 distinct variants involving the hemoglobin gene cluster). The authors emphasize that “comprehensive genetic screening for hemoglobin variants is crucial for prevention and treatment of these conditions”. However, “In China, the conventional methods of genetic testing … typically detect only the 24 hotspot variants commonly found in the Chinese population.” PacBio sequencing overcomes this limitation by “comprehensively encompass[ing] the full spectrum of known structural variations, single nucleotide variants (SNVs), and insertions/deletions (InDels) involving the HBA1, HBA2, and HBB gene clusters.” The study describes a PacBio-based population genetic screening program of 11,019 individuals in Guangxi, China. It also included and in silico structural predictions using Alphafold2 to assess the functional impact of rare variants. In total, researchers identified 165 hemoglobin variants, including two ultra-rare (0.0045%) variants at the same genetic locus, with different impacts on hemoglobin structure. “the identification of the variant expanded the mutational spectrum of the HBB gene in the Chinese population. It should be declared that this variant had not been included in the routine screening panels in most clinical settings in China”. They conclude that “SMRT sequencing has emerged as a reliable method for preconception screening and prenatal testing of hemoglobinopathies.” The authors suggest that integrating this approach into screening programs for newborns in high-incidence areas “could enhance early diagnosis, enable personalized treatment, support informed decision-making, and ultimately improve public health outcomes for these conditions.”
Conclusion: Finding these rare variants matters. Many aren’t included on standard screening panels, which means families could miss crucial information about their health and risk of developing diseases like thalassemia. By uncovering what other technologies miss, PacBio sequencing can help empower answers for families in high-risk regions.
In this preprint, authors from Israel, Broad, UPenn accomplish “the first single-cell splicing atlas of human CD8⁺ T cells, capturing dynamic isoform programs across activation and subset differentiation.”
Key highlights:
“Immune checkpoint blockade has transformed cancer therapy, yet many patients fail to respond”. The authors note that “Alternative splicing dramatically diversifies the T cell proteome, but the functional roles of most isoforms remain unknown.” “By integrating PacBio long-read sequencing for precise isoform resolution … we generated a high-resolution atlas of dynamic RNA expression and splicing in activated CD8 T cells, offering a detailed view of splicing patterns at single-cell resolution”. This approach “revealed distinct splicing footprints that refine conventional transcriptomic states and highlight receptor families with isoform-level regulation.” “~50% of DEIs [differentially expressed isoforms] reflected isoform-specific regulation without corresponding changes in overall gene expression”, and “splicing profiles more accurately reflected differentiation trajectories and provided additional resolution for both T cell subset delineation and pathway-level analyses.” To explore functional relevance, authors “developed SpliceSeek, a CRISPR-based pooled screening platform that perturbs splice sites to redirect isoform usage.” Using this platform, “we uncovered isoform-specific immune checkpoints whose perturbation enhanced effector function and tumor control, including the LRRN3-203 isoform, which augmented cytokine secretion and antitumor immunity in mice models.” “Our work establishes a generalizable framework: starting from isoform discovery to mechanistic characterization and ending with in vivo validation, which enables the development of isoform-selective immunotherapies.”
Conclusion: This study shows the power of combining HiFi long-read sequencing with single-cell resolution to map isoform usage in human CD8⁺ T cells. By capturing dynamic splicing programs that short-read methods often miss, the team not only redefined T cell states, but also uncovered novel immune checkpoints with therapeutic potential. This work lays the foundation for isoform-selective immunotherapies that are anchored in discovery, validated in vivo, and guided by a generalizable single-cell framework.
Ready to make discoveries of your own?
September’s publications remind us that HiFi sequencing is a lens into biology that other methods can’t provide. The researchers featured here are solving technical problems and expanding what’s possible in research, population health, and therapeutic discovery.
As these studies show, long-read sequencing continues to heighten our understanding of complex genetic landscapes in rare disease applications, cancer labs, and global screening programs.
Stay tuned for next month’s roundup to see how scientists around the world are putting HiFi sequencing to work in their own fields.
Ready to explore what HiFi can do in your lab? Let’s get started.