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Targeted long-read sequencing of native DNA for genetic disease diagnostic and screening research

Clinical Genetics and Therapeutics
  • Primary Categories:
    • Clinical Genetics
  • Secondary Categories:
    • Clinical Genetics
Introduction:
Short tandem repeats (STRs) are DNA sequences composed of repetitions of 2 – 6 bp motifs. Expansions of STRs are the cause of over 60 monogenic diseases, including Huntington’s disease, fragile X syndrome, and amyotrophic lateral sclerosis. In addition to their length, the pathogenicity of these STRs can be impacted by sequence composition, methylation status and mosaicism. One such example is the FMR1 repeat whose CGG repeat expansions are typically hypermethylated and where AGG interruption sequences can stabilize the repeat. Detecting all the characteristics associated with pathogenic repeat expansions traditionally required multiple assays, however high-accuracy long-read sequencing of unamplified DNA can resolve all these features in a single assay.

Methods:
We performed the PacBio PureTarget assay on a set of Coriell DNA samples with known repeat expansions. We sequenced the resulting libraries on multiple PacBio instruments and chemistries: Revio with and without the latest SPRQ chemistry, and the latest benchtop sequencer, Vega. Analysis of the samples was performed using the PureTarget workflow on SMRT Link. This workflow allows for the intuitive evaluation of all PureTarget samples in a single analysis.

Results:
The combination of the PureTarget assay and analysis solutions allows for accurate repeat genotyping and repeat expansion detection. The TRGT visualizations provided in the SMRT Link workflow allow for the interpretation of sequence composition, methylation status and allele mosaism. We also demonstrate the ability to define and resolve structurally complex repeat alleles (as needed for RFC1), as well as detect methylation.

Conclusion:
The PureTarget assay and TRGT software suite in SMRT Link allows for a comprehensive look at repeat expansions. This sample-to-answer solution allows for the detection and visualization of repeats with, and without mosaism, their sequence composition and their methylation status in one system.

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