A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings

Area of Science:

• Genomics
• Bioinformatics
• Molecular Biology

Background:

• Haplotype reconstruction aids disease risk interpretation but faces clinical cost and technical barriers.
• Second-generation sequencing (short reads) relies on indirect statistical haplotyping.
• Third-generation sequencing (long reads), like Oxford Nanopore Technologies (ONT), enables direct haplotyping with fewer limitations.

Purpose of the Study:

• To develop and validate a streamlined workflow for variant calling and phasing using ONT long-read sequencing data.
• To assess the feasibility of direct haplotyping in a clinically relevant 12-kb region of the APOE locus.
• To establish robust standards for variant phasing in ONT-based target resequencing.

Main Methods:

• Utilized ONT long-read sequencing on amplicons of the APOE locus.
• Developed a proof-of-concept workflow for variant calling and phasing.
• Evaluated different ONT read set sizes and bioinformatics software combinations (BWA/Minimap2, HapCUT2).

Main Results:

• Demonstrated reliable single-nucleotide variant (SNV) calling and phasing from as few as 60 ONT reads.
• Identified optimal conditions (600 reads, specific software) for full haplotype reconstruction including indels when pre-identified.
• Showcased the efficiency of ONT data for analyzing multiple samples in parallel.

Conclusions:

• Established a rapid, inexpensive, and implementable workflow for variant phasing using ONT long reads.
• The workflow facilitates direct haplotyping in the APOE locus, applicable to clinical settings and diagnostic testing.
• Overcomes limitations of traditional sequencing methods for haplotype reconstruction.