Validated DNA isolation method ensuring successful long-read sequencing of cattle semen genome

Area of Science:

• Genomics
• Molecular Biology
• Animal Science

Background:

• High-quality DNA is crucial for genomic studies, but isolating it from mammalian spermatozoa is challenging due to tightly packed DNA and robust cell membranes.
• Existing methods often struggle with sperm DNA extraction, potentially leading to fragmentation and impacting downstream sequencing applications like long-read sequencing.

Purpose of the Study:

• To describe a novel, optimized protocol for isolating high molecular weight (HMW) DNA from bovine spermatozoa preserved in commercial semen straws.
• To enable efficient and reliable DNA extraction from a challenging cell type for advanced genomic analyses, including long-read sequencing.

Main Methods:

• A modified salting-out DNA extraction method was employed, specifically adapted for bovine semen straws.
• The protocol incorporates a cleaning step to remove semen diluents and preservatives.
• Specialized chaotropic and reducing agents were used to disrupt sperm cell membranes and disulfide bonds, while minimizing DNA fragmentation through gentle handling and avoiding spin columns.

Main Results:

• The developed method successfully isolated high molecular weight DNA from bovine semen straws.
• Average DNA fragment size was approximately 49 kb, with a range of 25–85 kb, confirmed by femto pulse profiles.
• Over 80 semen samples were processed, yielding DNA suitable for Continuous Long-Read (CLR) sequencing on the PacBio SequelII platform.

Conclusions:

• This protocol provides an effective means to obtain high-quality HMW DNA from bovine spermatozoa, overcoming previous technical hurdles.
• The isolated DNA is suitable for long-read sequencing, facilitating comprehensive genomic studies including the detection of large structural variations in cattle.
• The method's robustness and efficiency make it valuable for bovine genomics research and breeding programs.