PacBio HiFi sequencing technology continues to be the tool of choice for genomics professionals working at the forefront of discovery, enabling them to pursue new avenues of exploration across diverse domains of biology.
In this edition of our Powered by PacBio blog series, we highlight scientific papers from the month of June 2024. These compelling publications highlight the power of PacBio sequencing to outperform other technologies for single-cell RNA-seq analyses, reveal segment duplications to better understand human disease, evolution and diversity, achieve species-level resolution, and more.
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Kinnex single-cell RNA-seq Population sequencing Gene regulation Metagenomics
Kinnex single-cell RNA-seq
In this paper, researchers from China performed both PacBio Kinnex & ONT sequencing “with 10x genomics scRNA-seq and compared them to the benchmark NGS using exact the same cDNA libraries of different sample sizes”:
Findings included:
- Long-read scRNA-seq “accurately captures all cell types”, “generated more consistent gene expression profiles compared with NGS”, “performed better than NGS in cell annotation with a small cell sampling size”, and “more cell type-specific molecules could be identified in a larger cell sample size”
- “PacBio identified a greater amount of cell type specific genes and isoforms” and “could specify more allele-specific transcripts”
- “PacBio demonstrates superior performance in discovering novel transcripts” and “novel isoforms identified using PacBio data were more accurate”
Conclusion:
“Attribute to higher sequencing quality of PacBio, it outperforms ONT in accuracy of novel transcripts identification and allele-specific gene/isoform expression.”
Population sequencing
Structural polymorphism and diversity of human segmental duplications
In this preprint, HiFi reveals segment duplications (SDs) unresolvable by short reads. This missing info is essential for understanding human disease, evolution and diversity. Researchers from HGSVC, UW, Altos Labs, JAX, CMKC conducted a study including a “population genetics survey of SDs by analyzing 170 [all HiFi] human genome assemblies where the majority of SDs are fully resolved using long-read sequence assembly.”
Key findings:
- “Notwithstanding their importance, understanding the genetic diversity of these more complex regions of the genome has been challenging.”
- “The development of PacBio HiFi (high-fidelity) sequencing technology and associated assembly algorithms has meant that most SD regions can be fully sequence resolved at the haplotype level.”
- We identified 173.2 Mbp of duplicated sequence (47.4 Mbp not present in the telomere-to-telomere reference) distinguishing fixed from structurally polymorphic events.
- “African genomes harbor significantly more intrachromosomal SDs and are more likely to have recently duplicated gene families with higher copy number when compared to non-African samples.”
- “A comparison to a resource of 563 million full-length Iso-Seq reads identifies 201 novel, potentially protein-coding genes corresponding to these copy number polymorphic SDs.”
Conclusion:
Understanding the genetic diversity of more complex regions of the genome (i.e., SDs) and expressed transcript isoforms therein are essential to understanding human disease, evolution and diversity. Using HiFi WGS and Kinnex uniquely resolves regions in the genome and transcriptome that are not accessible with any other technologies.
Gene regulation
A haplotype-resolved view of human gene regulation
In this preprint, a team of scientists from UW, Benaroya Res Inst at Virginia Mason Franciscan Health WA, Talus Bioscience WA, Seattle Children’s, Mayo, and Brotman Baty found that “Fiber-seq Inferred Regulatory Elements (FIREs) enable the accurate quantification of chromatin accessibility across the 6 Gbp diploid human genome with single-molecule and single-nucleotide precision.”
Key takeaways:
- “We find that cells can harbor >1,000 regulatory elements with haplotype-selective chromatin accessibility (HSCA) and show that these elements preferentially localize to genomic loci containing the most human genetic diversity, with the human leukocyte antigen (HLA) locus showing the largest amount of HSCA genome-wide in immune cells.”
- “We uncover HSCA elements with sequence non-deterministic chromatin accessibility, representing likely somatic epimutations”
- Using “paired haplotype-phased long-read transcript data [Kinnex]”, “This approach also disentangles gene regulation across the human inactive and active X chromosomes with nucleotide precision.”
Conclusion:
“FIRE enables the accurate de novo construction of a cell’s chromatin-accessible landscape directly using long-read sequencing data, enabling the advent of synchronous multi-omic long-read sequencing.”
Metagenomics
Microflora Danica: the atlas of Danish environmental microbiomes
In this preprint, scientists from Denmark, Australia, and Austria, conducted a study where HiFi sequencing was used for rRNA operon sequencing for 449 (multiplexed in pools of 92 samples) microbiome samples (14.9 million bacterial (median 4,528 bp, containing both 16S and 23S) and 13.4 million eukaryotic rRNA operon sequences (median 4,035 bp, containing both 18S and 28S)). “This dataset is an order of magnitude larger than the current most comprehensive database SILVA 138.1”, and provides “an unprecedented resource and the foundation for answering fundamental questions underlying microbial ecology: what drives microbial diversity, distribution and function.”
Conclusion:
Accurate, full-length sequencing (16S-ITS-23S, 18S-ITS-28S amplicons) provides high-quality, species-level resolution, improving the understanding of microbial diversity, distribution, and function and accurate, high-throughput long-amplicon sequencing with Kinnex 16S sequencing enables comprehensive microbial community characterizations
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These recent publications exemplify the versatility and power of PacBio sequencing. From tackling challenges associated with single-cell RNA-seq analyses to revealing segment duplications, PacBio technology is enabling scientific pioneers to make transformative breakthroughs like never before.
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