Abstract
The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.
Highlights
When a group of genetic variants is inherited together on a chromosome from one parent because of their genetic linkage, it defines a haplotype [1]
To address the lack of guidelines for users wishing to perform haplotype phasing based on Oxford Nanopore Technologies (ONT) sequencing reads, we presented a workflow for single-nucleotide variant (SNV) calling and haplotype phasing of variants localized in a region of interest
An average of 99.99% MinION PASS reads mapped properly to the target region and 98.07% of these reads spanned the entire 12-kb target
Summary
When a group of genetic variants is inherited together on a chromosome from one parent because of their genetic linkage, it defines a haplotype [1]. Second-generation NGS technologies, such as Illumina sequencing by synthesis, produce short reads (up to 300 bp) that usually span no more than a single variant, and allow only indirect haplotype reconstruction based on statistical inferences from population genotyping data. Several library preparation methods that preserve haplotype information in short-read sequencing data have been proposed These include mate-pair libraries, the single-cell DNA template strand sequencing (Strand-seq) [20], the proximity-ligation libraries of the Chicago [21], and Chromosome Conformation Capture (Hi-C) [22]. The method exploits available and validated software and requires only a modest number of ONT reads, enabling efficient haplotype phasing at low costs in clinical settings
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