In genomics, DNA is often compared to a cookbook, filled with recipes that guide the cells in your body. But where do tandem repeats fit into this analogy? Think of them as a simple instruction, like adding a pinch of salt to your soup. It’s fine when done once or twice, but imagine repeating it a thousand times—it could spoil the entire dish.
Tandem repeat expansions, repeating units of specific DNA sequences, can become pathogenic when they grow too long and have been linked to various diseases, including cancers. These expansions reside in the “dark regions” of the genome, areas that older sequencing technologies often struggle to read accurately.
This is where PacBio HiFi sequencing makes a difference. HiFi sequencing offers the length and accuracy needed to confidently navigate these challenging, repetitive regions. In this blog, we’ll explore the latest groundbreaking research that leverages the power of HiFi long reads to uncover repeat expansions and identify the mutations driving degenerative diseases.
HiFi sequencing shines a light on dark regions of the genome
HiFi sequencing combines the accuracy of traditional short reads with the long read lengths needed for comprehensive variant detection. With exceptional accuracy, uniform coverage, and nonexistent GC bias, HiFi sequencing delivers the ability to detect virtually all genomic variants.
While HiFi sequencing is known for the major benefits it brings to whole genome sequencing, it is also breaking ground in targeted sequencing applications, such as the aforementioned “dark” regions and tandem repeats. Already, PacBio has partnered with leading researchers to develop predesigned panels that target dark regions of the genome and genes that can have an impact on medication safety and efficacy, namely the Twist Alliance Dark Genes panel and Long-read Pharmacogenomics panel, respectively.
PacBio has now leveled up its set of targeted gene panels with the release of the PureTarget repeat expansion panel, designed to uncover critical insights into neurological disorders. PacBio also developed a tool for calling and visualizing tandem repeats called TRGT and TRVZ, which can be used to analyze both whole genome and targeted data1.
PureTarget long-read sequencing panel powers clinical research in neurological disorders
In March of 2024, PacBio announced the release of the PureTarget repeat expansion panel. Containing 20 clinically-relevant genes, this panel targets difficult-to-sequence tandem repeats, regions that were previously considered “dark” but are now accessible thanks to the accuracy of HiFi sequencing. These advancements are making significant strides in understanding diseases like ataxia, Huntington’s disease, amyotrophic lateral sclerosis (ALS), and Fragile X Syndrome.
| Disease | Targets |
|---|---|
| Spinocerebellar ataxia | ATN1, ATXN1, ATXN2, ATXN3, ATXN7, ATXN8, ATXN10, CACNA1A, PPP2R2B, TBP |
| Friedrich's ataxia | FXN |
| CANVAS disease | RFC1 |
| Fragile-X disease (FXS) | FMR1 |
| Huntington's disease | HTT |
| Myotonic dystrophy | DMPK, CNBP |
| Amyotropic lateral sclerosis (ALS), frontotemporal dementia (FTD) | C9orf72 |
| Fuchs endothelial corneal dystrophy | TCF4 |
| Spinal bulbar muscular atrophy / Kennedy's disease | AR |
| Oculopharyngeal muscular dystrophy | PABPN1 |
The PureTarget panel is based on an extensively updated and optimized version of the older No-amp method2. PureTarget utilizes an advanced CRISPR-Cas9 enrichment method that ensures high accuracy by preserving native DNA, which retains essential epigenetic signatures including methylation at CpG sites. This “PCR-free” approach reduces errors and has less size bias compared to traditional PCR methods, making it an essential tool in modern genomic research. This panel also consolidates multiple targets and enables sample multiplexing, so researchers can quickly generate a lot of data for their targets of interest.
Advancing research on degenerative eye diseases with PureTarget
One area where the PureTarget panel has made a profound impact is in the study of tandem repeat-associated degenerative eye diseases. This panel was central to the research on Fuchs Endothelial Corneal Dystrophy (FECD) led by Dr. Alice Davidson at University College London and presented at the 2024 European Society of Human Genetics Conference. FECD is a common, age-related degenerative condition that causes swelling of the cornea and results in blurry or cloudy vision. A cornea transplant is currently considered the best option for care, but given failure rates and increasing demand for donor tissue, new therapies are urgently needed to replace this “last-resort” option.
It has previously been discovered that 81% of people with FECD have a triplet-repeat (CTG18.1) within the TCF4 gene2,3. And as is the case with most tandem repeat disorders, a repeat length above a certain threshold corresponds with presentation of the disease, often with increasing severity.
Early research with PacBio targeted sequencing demonstrated that this CTG18.1 repeat is unstable in blood4, but additional questions about the mechanism of how this repeat influences FECD remained. With the recent release of the PacBio PureTarget repeat expansion panel and its coverage of TCF4, the UCL scientists were able to streamline their protocol, reduce DNA inputs, increase multiplexing capacity, and accelerate progress on their research.
Using patient-matched leukocytes and cultured corneal endothelial cells (which are difficult to obtain in large quantities), the researchers discovered CTG18.1 repeats from 1000 to >5500 tandem repeats exclusively within affected eye cells and find that once expanded, CTG18.1 is highly dynamic and unstable in the diseased corneal endothelium. This finding suggests that the instability of these repeats may be driving the symptoms of FECD, offering a new direction for therapeutic development.
Serving as a model system for tandem repeat diseases that are more difficult to study, this research highlights how the PureTarget panel is enabling scientists to dive deeper into the genetic causes of degenerative diseases, offering hope for new treatments where there were once only questions.
Using PureTarget to unlock the genetics of C9orf72 in ALS and FTD
The need for a more comprehensive repeat expansion panel is especially apparent in the study of ALS, a devastating disease that leads to the progressive loss of muscle control. The lab of Dr. Marka van Blitterswijk at the Mayo Clinic is performing several projects utilizing this technology. Evan Udine, a Ph.D candidate in her lab, is leading studies focused on the C9orf72 gene, which is known to be associated with ALS and frontotemporal dementia (FTD). Their goal was to find a more efficient method to characterize C9orf72 repeats, moving away from the labor-intensive Southern blot method.
The team’s original exploration with No-Amp sequencing provided results that matched and even surpassed those obtained by traditional methods, revealing important correlations between repeat length and disease severity. Initial exploration of tandem repeats from blood and brain samples were on par with Southern blot analysis, and revealed that shorter C9orf72 expansions were associated with longer survival time5.
Interestingly, their Southern blotting studies revealed that when examining blood samples taken from the same individuals over time, repeat length can be stable in some samples, but dynamic in others. Similarly, they found that repeat lengths differed even within affected and unaffected family members6.
With the enhanced resolution of long-read sequencing data from the PureTarget panel and Kinnex kit for RNA sequencing, the researchers have observed improvements in read coverage and length in a few pilot studies. The lab has numerous ongoing projects utilizing this technology to capture C9orf72 repeat expansions in the blood and multiple brain regions that will reveal relevant clinical and pathological associations.
Their continued work with the PureTarget panel promises to further advance the research of C9orf72 repeat expansions related to ALS and FTD.
Combatting ALS with PureTarget and CRISPR gene editing
As research into ALS advances, the potential for therapeutic interventions becomes increasingly promising. Dr. Claire Clelland’s lab out of UC San Francisco is at the forefront of using CRISPR-Cas9 to edit disease-causing mutations of ALS/FTD. As evidence of this approach, a previous study has shown that even 10% editing of C9orf72 can lead to more than a 50% reduction in pathology in animal models7.
Initial research with PacBio targeted sequencing of C9orf72 demonstrated that HiFi sequencing is more accurate and sensitive than other methods, including Southern blots and gene-specific and repeat-primed PCR. HiFi sequencing also allowed for accurate phasing of the repeat expansion by separating maternally and paternally inherited copies of the gene into haplotypes8.
Accurate phasing is critical for gene editing research because it allows you to determine editing outcomes through the purity of clones. Sanger sequencing can yield unclear molecular signatures that, without phasing, are impossible to distinguish an impure clone from a single clone with different alleles.
Now with the adoption of the PureTarget panel, the Clelland lab has expanded its research to visualizing repeat mosaicism in the C9 cell line, in which different cells exhibit variable attributes of repeat expansions. The group has also been evaluating methylation with the PureTarget panel, which serves as an emerging avenue for understanding efficacy and safety in gene editing research.
The continued success of the PureTarget panel in this research points to a future where degenerative diseases like ALS might not just be understood but also treated with precision.
Unlocking new possibilities in molecular diagnostics with PureTarget and HiFi sequencing
The pioneering work being done with the PacBio PureTarget panel is opening new doors in research surrounding degenerative repeat expansion disorders. By enabling comprehensive genotyping of notoriously difficult regions of the genome, this tool is helping researchers uncover the genetic causes of degenerative diseases and, ultimately, offering hope for new treatments.
Join these innovators by learning more about how PureTarget and HiFi sequencing enable comprehensive genotyping of these notoriously difficult regions of the genome.
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References
- Dolzhenko, E., et al. (2024). Characterization and visualization of tandem repeats at genome scale. Nature Biotechnology, 1-9.
- Tsai, Y. C., et al. (2022). Identification of a CCG-enriched expanded allele in patients with myotonic dystrophy type 1 using amplification-free long-read sequencing. The Journal of Molecular Diagnostics, 24(11), 1143-1154.
- Wieben, E. D., et al. (2012). A common trinucleotide repeat expansion within the transcription factor 4 (TCF4, E2-2) gene predicts Fuchs corneal dystrophy. PLoS One, 7(11), e49083.
- Fautsch, M. P., et al. (2021). TCF4-mediated Fuchs endothelial corneal dystrophy: Insights into a common trinucleotide repeat-associated disease. Progress in retinal and eye research, 81, 100883.
- Hafford-Tear, N. J., et al. (2019). CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat. Genetics in Medicine, 21(9), 2092-2102.
- DeJesus-Hernandez, M., et al. (2021). Long-read targeted sequencing uncovers clinicopathological associations for C9orf72-linked diseases. Brain, 144(4), 1082-1088.
- Jackson, J. L., Finch, N. C. A., et al. (2020). Elevated methylation levels, reduced expression levels, and frequent contractions in a clinical cohort of C9orf72 expansion carriers. Molecular neurodegeneration, 15, 1-11.
- Meijboom, K. E., et al. (2022). CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. Nature communications, 13(1), 6286.
- Salomonsson, S. E., Maltos, A. M., et al. (2024). Validated assays for the quantification of C9orf72 human pathology. Scientific Reports, 14(1), 828.