DNA-pools targeted-sequencing as a robust cost-effective method to detect rare variants: Application to dilated...

Area of Science:

• Genetics
• Cardiology
• Molecular Biology

Background:

• Dilated cardiomyopathy (DCM) is a heart condition with genetic underpinnings.
• Genetic diagnosis of DCM often involves identifying rare, private pathogenic variants.
• Current genetic testing methods like Next Generation Sequencing (NGS) can be costly.

Purpose of the Study:

• To develop and validate a cost-effective NGS strategy for DCM genetic diagnosis.
• To assess the robustness of a pooled DNA sequencing approach for rare variant detection.
• To reduce the financial burden of genetic testing for DCM patients.

Main Methods:

• Implemented a Next Generation Sequencing (NGS) strategy using pooled DNA from 8 DCM patients per pool.
• Utilized Freebayes software to detect variants with an expected 1/16 allele frequency.
• Optimized variant quality parameters using data from 96 pooled samples and validated against simplex sequencing.

Main Results:

• The pooled sequencing method achieved 96% accuracy in identifying single nucleotide variants (SNVs) and insertions/deletions (InsDels) compared to simplex sequencing.
• Detected 100 pathogenic variants (ACMG class 4 and 5) in 384 DCM patients, primarily in known DCM-associated genes (TTN, MYH7, FLNC, TNNT2).
• Achieved a 4-fold cost reduction for rare variant detection.

Conclusions:

• An original pooled-sequencing NGS method accurately detects rare variants in DCM patients.
• This innovative approach offers a cost-effective solution for genetic diagnostics in rare diseases.
• The method demonstrates high accuracy and significant cost savings for DCM genetic screening.