Targeted long-read sequencing identifies missing disease-causing variation.

Area of Science:

• Genomics
• Molecular Diagnostics
• Genetic Medicine

Background:

• Many individuals with suspected genetic disorders remain undiagnosed after conventional genetic testing.
• Challenges include interpreting structural variants, identifying variants of unknown significance, and detecting single pathogenic variants in recessive conditions.

Purpose of the Study:

• To evaluate targeted long-read sequencing (T-LRS) as a tool for precise genetic diagnosis.
• To assess T-LRS's ability to detect diverse genomic aberrations, including structural variants and methylation differences, in individuals with unresolved genetic conditions.

Main Methods:

• Targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform was performed on 40 individuals.
• Computational targeting focused on up to 151 Mbp of sequence per individual.
• Analysis aimed to detect pathogenic substitutions, structural variants, and methylation differences.

Main Results:

• T-LRS successfully identified all genomic aberrations previously detected by clinical testing, including single-nucleotide variants, copy number changes, and repeat expansions.
• Complex structural rearrangements were precisely resolved in 8/8 individuals, altering clinical management in one case.
• In individuals with undiagnosed Mendelian conditions, T-LRS identified pathogenic or likely pathogenic variants in 6/10 and variants of uncertain significance in 2/10.

Conclusions:

• T-LRS is effective in identifying pathogenic structural variants and resolving complex rearrangements.
• This method accurately detects Mendelian variants missed by other technologies.
• T-LRS provides an efficient and cost-effective strategy for evaluating high-priority genetic regions and complex cases.