A refined amplification-free long-read sequencing method to interrogate TCF4 triplet repeats associated with Fuchs endothelial corneal dystrophy

Abstract

Purpose : We have previously demonstrated the application and utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (termed CTG18.1). Here we aim to improve on our previously published method to characterise CTG18.1 in a FECD patient cohort using a dual cutter CRISPR/Cas9 enrichment approach in combination with PacBio single molecule real-time (SMRT) long-read sequencing.

Methods : An amplification-free method using a dual CRISPR/Cas9 system, in combination with PacBio SMRT long-read sequencing on a Sequel II platform, was applied to target CTG18.1. FECD patient-derived gDNA samples comprising a diverse range of CTG18.1 allele lengths were analysed. A robust data analysis pipeline was devised and applied to effectively filter, align and interrogate CTG18.1 specific reads.

Results : Dual CRISPR/Cas9-guided SMRT sequencing of CTG18.1 provided accurate genotyping information and enabled us to characterise the levels of somatic mosaicism present for all samples analysed. Advantages of this refined protocol, when compared to our previous method, include the enrichment and sequencing of a larger region of genomic DNA flanking CTG18.1 to enhance the likelihood of encompassing informative polymorphic markers to phase CTG18.1 reads. Furthermore, the dual cutter CRISPR/Cas9-guided design, in combination with sequencing on the PacBio Sequel II System, also enabled enhanced multiplexing capacity and reduced genomic DNA input requirements compared with our original protocol.

Conclusions : CRISPR-guided SMRT sequencing provides a powerful approach to accurately interrogate disease-associated tandem repeat expansions at a nucleotide level and detect levels of instability that are often prevalent within somatic tissues. The refined method presented here offers enhanced phasing capabilities, reduction of the amount of DNA required and increased multiplexing capacity compared to previously published methods.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.