Long-range PCR and Nanopore sequencing for localisation and phasing variants: an end-to-end clinical application...

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Short-read sequencing has limitations in phasing distant variants and analyzing homologous regions.
• Accurate phasing of variants is crucial for identifying compound heterozygosity.
• Long-read sequencing offers improved haplotype analysis for complex genomic regions.

Purpose of the Study:

• To develop and validate a robust workflow for variant phasing and localization.
• To enable clinical implementation of long-read sequencing for genetic diagnostics.
• To overcome limitations of short-read sequencing in complex genomic analyses.

Main Methods:

• Optimized long-range PCR (LR-PCR) for targets up to 20 kb using four kits.
• Targeted Nanopore sequencing on Flongle flow cells with barcoded amplicons.
• Developed an in-house bioinformatics pipeline incorporating Clair3, WhatsHap, and HapCUT2 for variant calling and phasing.

Main Results:

• The UltraRun LongRange PCR Kit achieved 90% success for DNA amplification up to 22 kb.
• All tested heterozygous SNV pairs and small InDels (up to 21.4 kb apart) were phased with 100% concordance.
• SNV calling in low-mappability genes showed precision and sensitivity of 1; median chimeric reads were 2.80%.

Conclusions:

• Established a reliable and affordable clinical workflow for phasing variants up to ~20 kb.
• Enables variant localization in regions inaccessible to short-read sequencing.
• Integrated approach improves complex genetic finding resolution and variant interpretation in diagnostics.