On the heels of ASHG, we are proud to see researchers leading the way with HiFi sequencing as the tool of choice to make groundbreaking discoveries. We are encouraged by their hard work and all of the knowledge still left to uncover through advanced sequencing by PacBio.
In this edition of our Powered by PacBio blog series, we highlight scientific papers from the month of November 2024. Studies explore how innovative sequencing workflows are enhancing the understanding of hereditary hearing impairment, and emerging techniques are expanding our understanding of single-cell epigenetics. From ultra-low input DNA protocols that enable assemblies from microscopic organisms to national-scale efforts advancing digital karyotyping for rare diseases, these publications showcase how PacBio technology continues to drive innovation across a variety of genomic applications.
Jump to topic:
Rare and inherited disease | Hearing impairment | Single-cell epigenetics | Ultra low-input DNA
Rare and inherited disease
A national long-read sequencing study on chromosomal rearrangements uncovers hidden complexities
This timely study from researchers in Sweden formally published four days prior to the start of November, and leading into ASHG. Here, “Genomic Medicine Sweden-Rare Diseases has explored the utility of HiFi Revio long-read genome sequencing (lrGS) for digital karyotyping of SVs nationwide”, with authors noting that “Based on our results, we propose a 5-year plan to expand lrGS use for rare disease diagnostics in Sweden.”
Findings from the preprint included:
- “Clinical genetic laboratories often require comprehensive analysis of chromosomal rearrangements/structural variants (SVs)… it is imperative to locate the breakpoint junctions (BPJs) and to resolve the derivative chromosome structure. This task, however, often surpasses the capabilities of conventional short-read sequencing technologies.”
- For this study. “the Genomic Medicine Sweden Rare Diseases (GMS-RD) consortium validated HiFi Revio long-read whole genome sequencing (lrGS) for clinical digital karyotyping of SVs nationwide”
- Authors utilized high-quality long-read genome data obtained from clinical DNA samples– “samples originated from five out of seven university hospital regions in Sweden and the DNA was not necessarily collected using lrGS-optimized protocols”
- The study “established a national pipeline and a shared variant database for variant callingand filtering. The included validation samples cover a spectrum of simple and complex SVs including inversions, translocations and copy number variants.”
- “De novoassembly resulted in a limited number of contigs per individual (N50 range 6-73 Mb) clearly separating the two alleles in most cases, enabling direct characterization of the chromosomal rearrangements.”, and “de novo assembly-based genome analyses are comprehensive and allow for the characterization of SVs alongside other variant types, such as SNV and repeat expansions.”
- Authors noted, “In future studies we strongly suggest analyzing SV in the context of trios or extended families, that allow high quality personal de novoassemblies be generated from the parents. Such assemblies will give a more accurate analysis of SV formation and genome dynamics of repetitive regions. Our results shown here indicate that this type of assemblies may be produced from the DNA obtained by current routine diagnostic workflows.”
Jesper Eisfeldt, Adam Ameur, Felix Lenner, et al. A national long-read sequencing study on chromosomal rearrangements uncovers hidden complexities. Genome Res. 2024 Oct 29. doi: 10.1101/gr.279510.124.
Conclusion:
The increased solve rates, combined with the increased throughput and lower cost from Revio, make HiFi sequencing an attractive choice for large, national program scale projects. Through best-in-class WGS that outperforms short-read genomes even at 20x coverage with the new SPRQ chemistry on Revio, researchers can move their ‘kitchen sink’ of different tests to a universal, efficient end-to-end HiFi sequencing workflow.
Hearing impairment
In this preprint, researchers out of Taiwan complete “the first large-scale clinical investigation utilizing LRS technology for the genetic diagnosis of SNHI [Sensorineural hearing impairment]”
Key findings:
- “SNHI-related pathogenic STRC variants cannot be directly addressed by conventional NGS due to the complex genomic scenario derived from large genomic rearrangements and a highly homologous pseudogene.”
- For this study, authors “developed a comprehensive workflow that integrates the PacBio-based LRS approach with marker-mediated refinements to effectively address pseudogene contamination”, and “applied to analyze the STRC gene in a cohort of 100 unrelated Taiwanese patients diagnosed with SNHI of unknown genetic cause after first-tier NGS testing”
- Researchers note, “We identified bi-allelic STRC variants in 11 patients (11% [additional] diagnostic yield), including homozygous deletions, compound heterozygous deletions and conversions, and compound heterozygous SNVs and CNVs”, and “we detected STRC variants in 27 patients, with 81.6% of these variants occurring in patients with mild to moderate SNHI”
- Noting, importantly: “Our study highlights the diagnostic capabilities of LRS in detecting complex variants within the STRC and advancing our understanding of the genetic etiology of SNHI that remains unresolved by conventional NGS.
Conclusion:
With cost-effective and targeted (long-range PCR or PureTarget expansion panel) and WGS on the Revio system (now with SPRQ chemistry) or on the Vega sequencer, researchers around the world are increasingly applying HiFi sequencing to larger disease cohorts and building out comprehensive workflows from sample to answers.
Single-cell epigenetics
Deaminase-assisted single-molecule and single-cell chromatin fiber sequencing
In this preprint, researchers from UW, Seattle Children’s found “comprehensive characterization of protein occupancy and chromatin accessibility across entire chromosomes with single-nucleotide, single-molecule, single-haplotype, and single-cell precision”.
Highlights:
- The study starts by presenting “Deaminase-Assisted single-molecule chromatin Fiber sequencing (DAF-seq), which leverages a non-specific double-stranded DNA deaminase toxin A (SsDddA) to efficiently stencil protein occupancy along DNA molecules via selective deamination of accessible cytidines, which are preserved via C-to-T transitions upon DNA amplification.”
- Authors note that this enables “single-molecule footprinting at near single-nucleotide resolution, enabling the precise delineation of the regulatory logic guiding neighboring proteins to cooperatively occupy chromatin fibers.”
- Further noting it enables “the synchronous identification of single-molecule chromatin and genetic architectures – resolving the functional impact of rare somatic variants, as well as transitional chromatin states guiding haplotype-selective promoter actuation.”
- Authors also found that, single-cell DAF-Seq enables “ultra-long consensus reads and chromosome-scale genomic phasing in single cells”: since each ‘haplotype-strand’ DNA template has a unique deamination pattern (from the strand-specific occupancy pattern of proteins, stochastic deamination & heterogeneous nucleosome positioning), even genomic regions of identical sequence will result in unique sequence reads. The overlap of these unique reads can be used to generate very long “individual ‘consensus reads’ for each strand and haplotype of that cell”. Demonstrating “thousands of consensus reads >100,000 bp in length”, and that “scDAF-seq enables the accurate reconstruction of the chromosome-scale haplotype-phased diploid genome from a single cell”. “These single-cell ultra-long consensus reads enable the evaluation of single-cell genomic and epigenomic variation within the most complex regions of the genome and lay the groundwork for potentially assembling telomere-to-telomere genomes and chromatin epigenomes from single cells.”
- The study enables “the near complete chromatin epigenome of a human diploid single cell”: whereas typical single-cell ATACseq (10,000-20,000 paired-end reads per cell) only resolves ~0.01% of the mappable genome. “In contrast, scDAF-seq enables the genetic evaluation of 96% of each cell’s mappable haploid genome, offering the potential for single-nucleotide precise maps of protein occupancy within a single cell – a ~7,000-fold improvement in our understanding of a cell’s chromatin architecture.”
Conclusion:
Studies like this highlight the expanding potential of HiFi-based applications through Revio with SPRQ chemistry and Vega sequencing to push the boundaries of discovery, empowering researchers with enhanced functionalities, diverse assays, and groundbreaking insights on their systems.
Ultra low-input DNA
And lastly, as a fun way to round out the month, researchers from Norway published a study utilizing an MDA-based ultra-low input protocol (including specimen directly added to Qiagen REPLI-g without prior DNA extraction), demonstrating high-quality HiFi assemblies for half a C. elegans (as control) and for one microscopic (~200 mm) hairybelly invertebrate individual.
The results:
- The hairybelly is “the first of its phylum”.
- This finding resolves “long-debated phylogenetic position” and comparative analysis of Hox cluster.
Ready to kickstart breakthroughs of your own?
These studies demonstrate the growing impact of PacBio sequencing in solving some of the most complex challenges in genomics. From national initiatives in rare disease diagnostics to cutting-edge single-cell epigenomics and the assembly of genomes from ultra-low input samples, HiFi sequencing is enabling researchers to push scientific boundaries.
With exceptional accuracy, enhanced throughput, and expanding accessibility through options like Revio and Vega, PacBio technology empowers scientists worldwide to unlock transformative insights.
Ready to take your research further? Discover how PacBio can accelerate your next breakthrough.