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

Excessive repeat sequence expansion in the human genome causes neurodegenerative diseases. Conventional tandem repeat expansion detection methods often fail to amplify GC-rich repeat regions and cannot help in simultaneously detecting multiple regions. To overcome those limitations, we tested the PacBio PureTarget repeat expansion panel, a new target enrichment test without PCR amplification, and compared its performance with that of conventional repeat expansion detection methods, using clinical and reference samples.

We used the PacBio PureTarget repeat expansion panel, which targets 20 genes with clinically relevant repeat regions, to assess eight samples from the Coriell Institute for Medical Research (Camden) and six patient samples (previously tested for FMR1 repeat expansions via repeat-primed [RP] PCR). Data were analyzed using the tandem repeat genotyping tool.

For all samples tested, the long-read sequencing results showed 100% concordance with the RP-PCR or Southern blotting results. Discrepancies in repeat counts were observed in a few alleles, with a maximum difference of 157 motifs in DMPK. The method successfully quantified long repeats in FMR1, demonstrating its applicability.

The PacBio PureTarget repeat expansion panel, which uses targeted enrichment based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 and long-read sequencing, is a promising approach for repeat expansion detection, overcoming the limitations of conventional methods. This approach enabled parallel analysis of multiple candidate genes implicated in neurodegenerative diseases with overlapping clinical features, supporting its potential integration into future clinical diagnostic workflows.