MASTR-seq enables multiplexed analysis of short tandem repeats with sequencing

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• Over 60 human disorders stem from unstable short tandem repeat (STR) expansions.
• Characterizing these expansions is challenging due to repetitive sequences and cell-type-specific mosaicism.
• Existing long-read methods are low-throughput and expensive for broad application.

Purpose of the Study:

• To develop a cost-effective, high-throughput method for analyzing STR genotype and DNA methylation.
• To achieve accurate measurement of STR length and methylation at single-allele resolution.
• To overcome technical challenges in characterizing repetitive DNA sequences.

Main Methods:

• MASTR-seq (Multiplexed Analysis of Short Tandem Repeats with Sequencing) was developed.
• The method combines long-read sequencing, Cas9-mediated target enrichment, size selection, and PCR-free multiplexed barcoding.
• It enables pooling of 8-12 samples per sequencing run.

Main Results:

• MASTR-seq provides accurate STR length and DNA methylation measurements.
• The technique offers single-allele resolution for analyzing both normal and expanded STR tracts.
• It successfully quantifies CGG, GGGGCC (G4C2), and CAG STR tracts.

Conclusions:

• MASTR-seq is a scalable and cost-effective solution for high-throughput STR analysis.
• This method facilitates the study of repeat expansion disorders by enabling detailed characterization of STRs and their methylation.
• MASTR-seq overcomes limitations of previous techniques, making it suitable for diverse experimental and clinical applications.