Performance Evaluation of PacBio PureTarget for Multiple Short Tandem Repeat Expansion Detection

Area of Science:

• Genomics
• Molecular Biology
• Neuroscience

Background:

• Excessive repeat sequence expansion in the human genome is a known cause of neurodegenerative diseases.
• Conventional methods for detecting these expansions often struggle with GC-rich regions and cannot analyze multiple sites simultaneously.

Purpose of the Study:

• To evaluate the PacBio PureTarget repeat expansion panel for detecting repeat expansions.
• To compare its performance against established methods for repeat expansion detection.

Main Methods:

• Utilized the PacBio PureTarget repeat expansion panel, targeting 20 genes with known repeat regions.
• Assessed eight reference samples and six patient samples, some previously analyzed by repeat-primed PCR.
• Analyzed sequencing data using a tandem repeat genotyping tool.

Main Results:

• Achieved 100% concordance between long-read sequencing results and conventional methods (RP-PCR or Southern blotting).
• Observed minor discrepancies in repeat counts for some alleles, with a maximum difference of 157 motifs in the DMPK gene.
• Successfully quantified long repeats in the FMR1 gene, confirming the panel's capability.

Conclusions:

• The PacBio PureTarget panel, employing CRISPR/Cas9 and long-read sequencing, offers a promising alternative for repeat expansion detection.
• This method overcomes limitations of conventional techniques and allows parallel analysis of multiple genes relevant to neurodegenerative diseases.
• The panel shows potential for integration into future clinical diagnostic workflows for genetic disorders.