Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing

Area of Science:

• Genomics
• Molecular Biology
• Genetic Diagnostics

Background:

• Targeted DNA sequencing often requires amplification, which can fail with repetitive or high Guanine-Cytosine (GC) content regions.
• Repeat expansions in genes like huntingtin (HTT) are linked to human diseases but are challenging to analyze with standard PCR methods.

Purpose of the Study:

• To develop and validate an amplification-free targeted sequencing protocol for studying repeat elements.
• To analyze repeat expansions in the huntingtin (HTT) gene associated with Huntington disease.
• To create a robust, alignment-independent data analysis pipeline for repeat element quantification.

Main Methods:

• Utilized CRISPR/Cas9 for amplification-free targeted enrichment (No-Amp Targeted sequencing).
• Employed single molecule, real-time (SMRT) sequencing for high-resolution analysis.
• Developed a novel bioinformatics pipeline for analyzing repeat elements without reference genome alignment.

Main Results:

• The No-Amp Targeted sequencing method accurately quantified CAG repeat counts in 11 diagnostic blood samples, showing agreement with fragment analysis for all 22 alleles.
• The amplification-free approach enabled the study of somatic variability in repeat elements, free from PCR stutter artifacts.
• Successfully applied the method to analyze challenging repeat elements in the HTT gene.

Conclusions:

• No-Amp Targeted sequencing combined with the developed analysis pipeline provides an accurate and reliable method for studying difficult-to-analyze repeat elements.
• This approach overcomes the limitations of PCR-based methods, offering improved insights into repeat expansions and their associated diseases.
• The amplification-free protocol is particularly valuable for diagnostic applications involving repetitive DNA regions.