Resolving the chromatin impact of somatic variants with targeted Fiber-seq

The majority of somatic variants lie outside of protein-coding sequences, where their functional consequences are often incompletely understood owing to the challenges associated with identifying individual DNA molecules that harbor somatic variants, as well as co-identification of the chromatin impact induced by these somatic variants along the same DNA molecule. To address this, we developed targeted Fiber-seq, which leverages non-specific N6-adenine methylation of accessible chromatin and CRISPR-Cas9-assisted enrichment to achieve single-molecule, long-read genomic and epigenomic profiling across >100 kilobase loci with approximately 10x enrichment over untargeted sequencing. Using deep-coverage targeted Fiber-seq, we demonstrate the simultaneous assessment of variants and their impact on chromatin architectures at single-molecule resolution in two models of somatic variation. Specifically, we demonstrate that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated gamma-globin (HBG1/HBG2) promoters in primary human hematopoietic cells increases the chromatin accessibility of both the HBG1 and HBG2 promoters as well as neighboring regulatory elements. Overall, we demonstrate that somatic genetic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.