CFTR haplotype phasing using long-read genome sequencing from ultralow input DNA.

Area of Science:

• Genomics
• Genetic disease screening
• Molecular biology

Background:

• Newborn screening detects rare genetic diseases caused by recessive inheritance.
• Long-read genome sequencing (LRS) aids in identifying and phasing genomic variants.
• Developing LRS for low-yield DNA samples is crucial for broader applications.

Purpose of the Study:

• To evaluate the utility of long-read genome sequencing (LRS) for identifying and phasing pathogenic variants in low-yield DNA samples.
• To compare LRS with short-read sequencing for variant detection and genotype concordance.

Main Methods:

• High molecular weight genomic DNA was extracted from two cystic fibrosis patient samples (whole blood and dried blood spot).
• Library preparation and genome sequencing (30-fold coverage) were performed using 20 ng of DNA on PacBio Revio and Illumina NovaSeq platforms.
• Single-nucleotide variants, small indels, and structural variants were identified and analyzed.

Main Results:

• Genotype concordance between LRS and short-read sequencing was higher for single-nucleotide variants than for small indels.
• Both sequencing technologies accurately identified known pathogenic variants in the CFTR gene.
• PacBio read-based haplotype phasing successfully determined allelic phase and identified compound heterozygosity of pathogenic variants.

Conclusions:

• LRS facilitates haplotype phasing of rare pathogenic variants even with minimal DNA input.
• This LRS approach may eliminate the need for parental testing in genetic disease screening.
• The study highlights LRS's potential to shorten the time to diagnosis for genetic diseases.