Use of Genomic Enrichment and Long Read Sequencing to Investigate Variant Repeat Patterns of Myotonic Dystrophy Type 1 CTG Expansions

Abstract

Background: Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG expansion of more than 50 repeats in the 3-prime untranslated region of the DMPK gene. The expanded repeat is unstable and expands in the soma and the germline. The length of the repeats and the presence of variant interruptions modifies disease onset and severity. No current available diagnostic methods are able to detect these interruptions with high accuracy at a low cost. In addition, complex variant sequences within the repeat often are undetectable using these methods. Demystifying these sequences will provide a better understanding of how their presence affects disease progression.

Aim: To determine if Oxford Nanopore Technologies (ONT) long-read sequencing of multiple displacement amplification (MDA) enriched alleles can accurately detect repeat length and sequences in a subset of samples. the MDA enrichment is expected to generate sufficient coverage without the need for extra amplification.

Method: The non-disease allele was removed using a Samplix Xdrop Sort and the expanded allele was enriched by MDA. Four samples were library prepped by debranching and then repairing before either being PCR amplified then sequenced, or directly sequenced by long-read nanopore sequencing. Through a bioinformatic pipeline, the resulting enrichment was calculated, and the length and sequence deciphered.

Results: The resulting reads showed a high enrichment for the target region similar, if not better than, reported results. ONT was able to determine the length and sequence of the samples. However, non-CTG repeats were seen in the middle of the reads, possibly attributed to sequencing errors.

Conclusion: Further research is needed to optimize the library preparation steps and explore the efficiency of other long-read sequencing methods with MDA enrichment.