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

More than 60 human disorders are caused by unstable expansion of short tandem repeat (STR) tracts. These can exhibit cell-type-specific mosaicism in several repeat expansion disorders and remain difficult to characterize due to technical challenges intrinsic to highly repetitive sequences. Long-read approaches can measure STR length and DNA methylation on the same single molecule but are low-throughput and cost-prohibitive across multiple experimental conditions or patient samples. Here, we present MASTR-seq, multiplexed analysis of short tandem repeats with sequencing, for cost-effective, high-throughput, accurate measurement of STR genotype and DNA methylation at single-allele resolution. MASTR-seq couples long-read sequencing, Cas9-mediated target enrichment, size selection, and PCR-free multiplexed barcoding to increase on-target read proportion for 8-12 pooled samples in a single MinION flow cell. MASTR-seq quantifies tract length and DNA methylation status for CGG, GGGGCC (G4C2), and CAG STR tracts in normal-length and mutation-length samples.