G007 Short tandem repeats sequencing: methodological challenges and perspective for Huntington’s disease
Year: 2024
Type: conference-abstract
Abstract: Background Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding for the huntingtin protein. Recently, Genome Wide Association Studies (GWAS) have identified genomic variations, occurring both at the HTT locus and in genes mostly involved in mismatch repair pathways, to be associated with disease onset and progression. Such mutations correlate with modulations of somatic instability, which is now considered as the main driver of pathogenesis. Accordingly, the set-up of reliable sequencing-based methods for assessing somatic instability is of paramount importance. Aims In this work, we aimed to compare workflows for assessing somatic instability, by identifying strengths and drawbacks of each enrichment, sequencing and analysis method. Methods Based on research studies focusing on Short Tandem Repeats (STR) characterization available in the literature, we compared multiple enrichment methods coupled to various sequencing platforms and data analysis tools. We then performed a preliminary comparison on internal data, and discussed their accuracy in CAG sizing and sensitivity for rare alleles detection. Results We reported much higher enrichment for PCR-based methods, compared to CRISPR/Cas9 enrichment and adaptive sampling. Moreover, unlike long-read sequencing platforms, we described a decrease in sequencing quality at cycles above 300 for Illumina MiSeq. As a last point, we showed a preliminary comparison among sequencing-based methods on internal data, showing the impact of each step towards the most accurate STR characterization. Conclusions We anticipate that long-read sequencing of PCR amplicons incorporating UMIs may represent a valid alternative to PCR-free enrichment methods, providing highly accurate performances in terms of repeat length and rare alleles detection. Nonetheless, we recommend accurate optimization of PCR primers and conditions, as suboptimal number of PCR cycles may result in off-target products and inadequate number of PCR duplicates.
Institutions: University of Milan, Istituto Nazionale Genetica Molecolare
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4402374839