Challenges and Approaches to Genotyping Repetitive DNA

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Copy number variation (CNV) in repetitive DNA elements is substantial within species.
• CNV can influence complex traits like lifespan and disease susceptibility.
• Accurate quantification of CNV, especially for large repetitive arrays like ribosomal DNA (rDNA), remains a significant technical challenge.

Purpose of the Study:

• To assess the technical reproducibility of major copy number estimation methods for the ribosomal DNA (rDNA) array.
• To compare the performance of pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing for rDNA copy number quantification.
• To identify potential causes of error in common quantification techniques and provide best practice recommendations.

Main Methods:

• Evaluation of pulsed-field gel electrophoresis (PFGE).
• Assessment of droplet digital PCR (ddPCR).
• Analysis of Nextera-based whole genome sequencing (WGS) for copy number estimation.
• Comparison across model organisms (S. cerevisiae, C. elegans) and diverse copy number ranges.

Main Results:

• Nextera-based WGS exhibited high error rates in rDNA copy number estimation.
• Pulsed-field gel electrophoresis and droplet digital PCR demonstrated better reproducibility for rDNA copy number.
• High-confidence rDNA copy number estimates were generated for S. cerevisiae and C. elegans strains.

Conclusions:

• Standard Nextera-based WGS is not reliable for accurate rDNA copy number quantification due to high error rates.
• PFGE and ddPCR are more robust methods for estimating rDNA copy number variation.
• Further exploration into FISH-based methods may offer cellular-level copy number characterization.