HiFi sequencing continues to drive discoveries across research areas, from chromatin architecture to immune system diversity and gene therapy safety.
In the February 2025 edition of Powered by PacBio, we explore a new low-input chromatin conformation capture method, a deeper look at hidden variation in the IGHC locus, and unexpected contaminants in lentiviral vectors.
Let’s dive into how HiFi sequencing is shaping the future of genomics.
Jump to topic:
Chromatin Assembly | How HiFi is Transforming Genomics | Immunology | Gene Therapy
Chromatin Assembly
CiFi: Accurate long-read chromatin conformation capture with low-input requirements
In this preprint by UC Davis, USDA HI, Sanger UK, and PacBio, researchers find that they can now achieve “haplotype-resolved, chromosome-scale de novo genome assemblies with data from one sequencing technology, and, if desired, from a single sequencing run.”
Key takeaways:
- CiFi (Hi-C with HiFi) generates multiple chromosome-interacting segments per HiFi read. Standard 3C protocols followed by improved PacBio low-input protocol (Ampli-fi workflow) results in >500-fold improved efficiency compared to previous approaches (5x higher sequencing yield, >100-fold reduced input).
- The study successfully demonstrated haplotype-resolved connectivity across all length scales, including those exceeding 100 Mb.
- They achieved improved resolution of chromatin conformation in repetitive & low-complexity regions (e.g. segdups, centromeres).
- These findings confirmed that you can achieve single-tech, low-input, chromosome-scale assembly from single insects (mosquito, fly), including dissecting a single insect in half and running HiFi and CiFi libraries simultaneously on a single Revio SMRT Cell.
Conclusion:
Together, CiFi and HiFi WGS on the Revio system and Vega system offer an increasingly expanded suite of applications with higher accuracy and lower input requirements than other solutions.
How HiFi is Transforming Genomics
Long and accurate: How HiFi sequencing is transforming genomics
In this article, researchers from China present a comprehensive overview of HiFi sequencing applications across various fields, including genomics (assembly, variant calling, complex regions, tandem repeats, phasing), transcriptomics (isoforms, fusion genes), epigenomics, metagenomics and single-cell sequencing.
Highlights:
- “Recent developments in PacBio high-fidelity (HiFi) sequencing technologies have transformed genomic research.”
- HiFi “circumvents biases intrinsic to amplification-based approaches, enabling thorough analysis of complex genomic regions [including tandem repeats, segmental duplications, ribosomal DNA (rDNA) arrays, and centromeres] as well as direct detection of base modifications, furnishing both sequence and epigenetic data concurrently.”
- “HiFi sequencing’s ability to generate long, highly accurate reads has revolutionized the field of genome assembly.”
- “Variant calling using HiFi sequencing technology achieves the highest precision and recall across all variant categories.”
- “it enables a more precise reconstruction of complex transcripts, thereby improving the identification of fusion genes and uncovering previously undetected isoforms with unprecedented accuracy.”
- “HiFi transcriptome sequencing data not only marks a substantial leap forward in cancer research but also holds immense promise for enhancing clinical diagnostics in the field.”
Conclusion:
HiFi sequencing powers a wide range of applications, and researchers worldwide continue to see its transformative impact on genomics.
Immunology
In this preprint, researchers from U Louisville KY, Mt. Sinai, UW, Bar-Ilan U Israel, and VRC NIH have found “a new foundation for investigating IGHC [immunoglobulin heavy chain constant] germline variation and its role in antibodies function and disease.”
Key highlights:
- Researchers used HiFi “to build a high-quality IGHC haplotype/variant catalog from 105 individuals [from 1KGP] of diverse ancestry”, including “a high-throughput approach for targeted [probe capture] long-read IGHC locus sequencing and assembly.”
- Noting, “we observed tremendous diversity”: from many previously uncharacterized variants (SNVs, complex SVs, novel genes and alleles).
- “Of the 262 identified IGHC coding alleles, 235 (89.6%) were undocumented [in the IMGT database].” This paper represents a 235% increase of newly discovered alleles in the database.
- “73% of SNVs genotyped in our long-read data were not called by the 1KGP. These data indicate the additional work needed to better integrate missing variation in genetic association studies.”
- They found “significant population differentiation”, including hundreds of SNVs in African and East Asian populations, and an IGHG4 haplotype carrying specific coding variants uniquely enriched in East and South Asian populations.
- Stating “our work in this locus is only just beginning … to delineate functional variants of biomedical importance. We show here that the use of long-read sequencing offers a tractable solution to this challenge.”
Conclusion:
Short-read sequencing misses much of IGHC variation, leading to incomplete genetic association studies. HiFi sequencing fully captures structural and sequence diversity, enabling researchers to characterize antibody function, uncover functional variants linked to immune-related diseases, and enhance precision medicine with population-specific insights.
Gene Therapy
In this study, researchers from the UK used PacBio sequencing to identify “multiple aberrantly packaged nucleic acid species in full genome carrying and empty LV [Lentivirus] particles.”
Key highlights:
- LV vectors are “one of the most commonly used vectors for clinical gene therapy of inherited disorders such as immune deficiencies and cancer immunotherapy”, however “one of the most difficult challenges … is the large-scale production of highly pure vector stocks … there has been a recent investment in the United Kingdom to establish good manufacturing process (GMP)-licensed centers for manufacture and quality control.”
- “Comprehensive detail on the full nucleic acid content of LV is unknown, even though they have entered clinical trials.”
- This study used PacBio technology to characterize recombinant HIV-1 particles generated by human 293T packaging cells, finding “a range of nucleic acids not of HIV-1 origin”, both from exogenous viruses (Table 2) and human endogenous retrovirus (HERV) elements.
- “Our data suggest that further investigation of the origin of these nucleic acids and their potential side effects on target cells should be considered and propose that removal of these contaminants would be a valuable step in manufacturing safer recombinant LV particles.”
Conclusion:
Hidden contaminants in lentiviral vectors could trigger immune reactions, disrupt treatment efficacy, or lead to unforeseen complications in drug trials. HiFi sequencing technology more fully exposes these risks, revealing unintended viral elements that other methods miss. By ensuring the highest vector purity, sequencing with PacBio can help protect against risks, prevent costly trial failures, and accelerate regulatory approval for safer, more effective gene therapies.
Ready to make discoveries of your own?
This month’s research highlights how HiFi sequencing is enabling deeper insights into chromatin structure, uncovering hidden immune system variation, and improving gene therapy safety. These studies demonstrate the power of long-read sequencing to tackle challenges that short reads miss, driving progress across multiple fields.
Stay tuned for next month’s edition as we continue to feature the latest breakthroughs made possible with HiFi sequencing.
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