PacBio HiFi sequencing technology continues to offer industry-leading results that enable researchers to explore uncharted territory across a variety of biological fields. The exceptional accuracy of HiFi sequencing drives new genetic insights, accelerating progress in disease research, agriculture, and beyond.
In this edition of our Powered by PacBio blog series, we spotlight the latest scientific papers from September 2024, showcasing breakthrough studies that leverage HiFi sequencing. From untangling the complexities of disease-associated transcript isoforms in the retina, to improving germline testing for rare diseases, and enhancing crop breeding strategies through pan-genomics, these papers underscore how PacBio technology is shaping the future of genomic research.
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Clinical and Translational RID Agrigenomics Microbial
Clinical and Translational
In this study, researchers out of Radboud Netherlands, Italy, and Belgium utilize the exceptional accuracy and coverage of the Iso-Seq method “to obtain a comprehensive overview of the Usher syndrome-associated transcript isoforms present in the human neural retina.” As the authors note, Usher syndrome is an inherited retinal disease (IRD) “which involves 11 associated genes with transcripts up to 19.6 kb.”
Key findings:
- In addition to standard Iso-Seq, researchers “employed the Samplix Xdrop System for indirect target enrichment of cDNA” which “facilitated the successful capture and sequencing of ADGRV1 transcripts as well as the full-length 18.9 kb USH2A transcripts.”
- “We revealed a more intricate isoform landscape than previously understood”, “we identified novel isoforms and alternative splicing events across the 11 Usher syndrome-associated genes, with implications for diagnostics and therapy development.” For example, “several ASO-based splicing modulation therapies under development for Usher syndrome-associated RP [retinitis pigmentosa].”
Conclusion:
Iso-Seq is now scalable and cost-effective with Kinnex library prep kits and sequencing with the Revio system. It offers exceptional precision in capturing full-length transcripts, especially in complex tissues like the retina, where alternative splicing is key. Unlike short-read sequencing, Iso-Seq provides comprehensive identification of transcript isoforms, which is critical for studying diseases such as Usher syndrome.
RID
HiFi long-read genomes for difficult-to-detect clinically relevant variants
In this study, researchers from Radboud U & Maastricht U Netherlands and PacBio find that “HiFi genomes may be a more attractive first-tier, generic assay for germline testing in rare diseases”.
Highlights:
- The study notes that “30x HiFi genomes for 100 samples with 145 known clinically relevant germline variants that are challenging to detect using short-read sequencing and necessitate a broad range of complementary test modalities in diagnostic laboratories.”
- Researchers “identified 93% of pathogenic variants that are most challenging to detect using short-read technologies.”
- Finding that “89% of all automatically called variants could also be identified using 15-fold coverage … possibly enhancing cost-effective diagnostic implementation.”
Conclusion:
“Our results show that LRS has the potential to be implemented as a first-tier diagnostic workflow for germline testing, potentially replacing all current tests for diagnosing individuals with rare disease.”
Agrigenomics
Solanum pan-genomics and pan-genetics reveal paralogs as contingencies in crop engineering
In this paper, researchers from CSHL, JHU, Boyce Thompson NY, Colombia, Uganda, Canada, UK, Mount Holyoke MA and Cornell use HiFi sequencing to unlock hidden gene complexity to drive breeding precision. Their work resulted in “establishing a pan-genome of the crop-rich genus Solanum and integrating functional genomics and genetics.”
Highlights included:
- HiFi-based, chromosome-scale references for 22 species, including 13 indigenous crops (average QV>53, average N50=65.8Mbp)
- Detailed pan-genome analysis, including a complex landscape of gene duplications (“hundreds of global and lineage-specific gene duplications exhibited dynamic evolutionary trajectories in paralog sequence, expression, and function, including among members of key domestication gene families”), core and dispensable genes, fate of retained paralogs with multi-tissue transcriptomics, and lineage-specific paralog diversification and compensatory relationships
- Analysis of additional 10 cultivars of African eggplant revealed that the “loss of an ancient redundant paralog of the classical regulator of stem cell proliferation and fruit organ number … was compensated by a lineage-specific tandem duplication.”
Conclusion:
“Paralog diversifications over short evolutionary periods are critical yet underexplored contingencies in trait evolvability”, and “demonstrating the value of resolving these underexplored contingencies as we strive to improve indigenous crops for local and climate change adapted agriculture.”
High-quality genome assembly with HiFi provides deeper insights into gene structure of important crop species. By accurately capturing complex gene variants, researchers can streamline breeding programs and genome-editing strategies, reducing trial-and-error experiments. The ability to transfer these genetic insights across related crops improves the predictability of genotype-to-phenotype outcomes, reducing the costs associated with failed breeding attempts and allowing cross-species trait selection. Understanding these traits facilitates advancements in both crop diversity and resilience which are crucial to protecting global food systems.
Microbial
In this study, researchers from the Todd Michael lab and Rob Knight lab et al. at Salk Institute, UCSD, Baylor, and WASHU published the first head-to-head comparisons of PacBio, ONT, ILMN, and TELL-Seq on generating MAGs from longitudinal pediatric microbiome samples.
Key findings:
- PacBio sequencing generates 2.4x more cMAGs (complete metagenome-assembled genomes) than ONT, with a lower cost per cMAG, and identifies the most microbiome associations with environmental and undernutrition variables.
- PacBio sequencing consistently outperforms ONT in key metrics: read quality, mean/median read lengths, and N50 read lengths, with higher overall accuracy and data quality. While ONT had a higher max read length, PacBio produced 2.43x more cMAGs on average at every data depth.
- PacBio cMAGs show significantly higher completeness and lower contamination than ONT, ensuring superior quality, with PacBio metaMDBG assembly generating nearly the same number of cMAGs as SR using just 1.4% of the data.
- Illumina SLR (TELL-Seq) did not yield any cMAGs across the 625 Gbp of generated data.
- PacBio cost per cMAG ($16) beats ONT ($25) and SR methods (up to $237), with the fewest unclassified reads, proving its efficiency and accuracy.
- PacBio detected the most significant associations (14 out of 26) and improved taxonomic profiling classification rates by 68%, outperforming other platforms in sensitivity and reproducibility.
*Note: This study utilized SMRTbell prep kit 3.0, whereas using HiFi prep kit 96 for metagenome assembly would further drop the cost per cMAG.
Conclusion:
HiFi metagenome sequencing delivers the most accurate and cost-effective long-read method for generating complete cMAGs, outperforming ONT, Illumina SR, and TELL-Seq (Illumina’s synthetic long read technology). It excels in detecting significant metadata associations and offers the highest taxonomic classification rates, making it ideal for challenging, uncultured metagenome environments, benchmark studies, and database reference genome creation.
Ready to kickstart breakthroughs of your own?
From decoding the intricate transcript isoforms in the human retina for improved understanding of inherited retinal diseases, to enhancing the detection of rare germline variants that are often missed by other methods, PacBio technology is driving major advances in genomics. Its impact extends beyond human health, where uncovering gene duplications in crop species is transforming the way we approach breeding and crop resilience.
Now, with flexible financing options and collaborations with certified service providers, PacBio sequencing is more accessible than ever for research teams of any size. Ready to unlock the next level of discovery in your work?