Nanopanel2 calls phased low-frequency variants in Nanopore panel sequencing data

Area of Science:

• Genomics and Bioinformatics
• Molecular Diagnostics

Background:

• Clinical decision-making relies on accurate detection of somatic variants and their haplotypes.
• Short-read sequencing has limitations in haplotype phasing due to read length, hindering detection of co-occurring variants.
• Long-read sequencing offers direct phasing but has been limited by high error rates.

Purpose of the Study:

• To develop a robust variant caller for Nanopore panel sequencing data.
• To enable accurate haplotype calling and somatic variant detection using long-read sequencing.
• To establish a fast, accurate, and cost-effective diagnostic workflow.

Main Methods:

• Developed Nanopanel2, a variant caller operating directly on base-called FAST5 files.
• Utilized allele probability distributions and filters to distinguish true variants from false positives.
• Enabled direct phasing of variants on the same read for haplotype determination.

Main Results:

• Nanopanel2 accurately calls single nucleotide variants (SNVs) and insertions/deletions (INDELs) down to 1% and 5% variant allele frequency, respectively.
• The caller produces minimal low-frequency false positives (approximately 1 FP call with VAF<5% per kb amplicon).
• Achieved direct phasing of amplicon variants, overcoming limitations of short-read sequencing.

Conclusions:

• Nanopanel2 is the first somatic variant caller designed for Nanopore sequencing data.
• Enables accurate, rapid (under 48 hours), and affordable (approx. $10/sample) diagnostic workflows.
• Facilitates improved clinical decision-making through direct haplotype phasing and sensitive variant detection.