Variant phasing for antisense oligonucleotide design using adaptive sampling

Biography

Joy Goffena is a Research Scientist in Dr Danny Miller’s lab at the University of Washington. She specializes in applying Oxford Nanopore Technologies sequencing to advance genetic research and clinical diagnostics. Her work focuses on identifying causal genetic variants, optimizing lab workflows for genetic testing, and bridging research and clinical applications to enhance patient care and accessibility to cutting-edge genomic testing.

Abstract



Phasing variants is critical for the development of antisense oligonucleotide (ASO) therapies, as common variants on the same haplotype as a pathogenic variant are often targeted for knockdown. Long-read sequencing offers extended read lengths that facilitate accurate haplotype resolution. However, the cost and throughput requirements can be prohibitive. To address these challenges, we used adaptive sampling (AS) to selectively enrich relevant regions for phasing of candidate target variants.

We performed AS on an Oxford Nanopore Technologies PromethION for 19 different genes across 27 samples derived from four different sources: previously extracted DNA (n = 8), blood (n = 6), assisted saliva (n = 10), and fibroblasts (n = 4). Approximately 5 Mbp of genomic space was targeted per sample, including control regions on the X chromosome and autosomes. Typically, a single sample was run for 24 hours before washing and either loading a new sample or storing the flow cell. Variant calling, phasing, and annotation were performed using a custom pipeline. Target genes of interest were manually evaluated to ensure complete phasing of variants across the entire gene.

Separately, we performed AS of six barcoded samples on a single PromethION Flow Cell with two washes and reloads. We recovered an average of 58× depth of coverage across all samples, approximately eight times the coverage over whole-genome background levels. There was markedly lower enrichment using DNA derived from saliva (four times the background coverage) than the other three sample types (10 times the background). The N50 of reads mapping to target regions ranged from 11 kbp for saliva, 23 kbp for blood- and fibroblast-derived DNA, and 31 kbp for previously extracted DNA.

Barcoding of six blood- or fibroblast-derived DNA samples resulted in approximately 14× times the background coverage, with a read N50 of 13 kbp. AS enabled accurate identification of known pathogenic variants in all cases and successful phasing of all SNV or indel variants within the gene of interest to single, contiguous phase blocks.

Our results demonstrate that AS on the Oxford Nanopore PromethION provides a robust, flexible approach for high-resolution haplotyping of clinically relevant variants. Despite modestly lower enrichment from saliva-derived DNA, we achieved sufficient depth and read length to accurately phase all pathogenic variants within the targeted genes across 27 samples from diverse sources.

This streamlined workflow, coupled with the ability to reuse flow cells, significantly lowers both cost and labor while maintaining the accuracy required for allele-specific antisense oligonucleotide design. Moving forward, the integration of targeted, haplotype-resolved sequencing into standard clinical and research pipelines holds promise for accelerating the development of precision therapies.