Tissue-specific DNA isolation from dissected millipedes for nanopore sequencing.

Area of Science:

• Genomics
• Molecular Biology
• Entomology

Background:

• The class Diplopoda (millipedes) is diverse, yet few species have sequenced genomes, limiting functional genetic studies.
• Characterizing genomes of non-model organisms is challenging due to complex morphology and difficulties in high-quality DNA extraction.

Purpose of the Study:

• To develop and detail a robust methodology for extracting high-purity DNA from millipede tissues for genomic analysis.
• To optimize DNA yield and quality from wild-caught specimens of *Cherokia georgiana*.

Main Methods:

• A detailed dissection protocol was employed to isolate tissues (legs, head, body) and exclude the digestive tract, minimizing microbial contamination.
• Sample homogenization techniques were optimized to enhance total DNA yield.
• DNA quality, concentration, and fragment length were assessed using spectrophotometry, fluorometry, and automated electrophoresis.
• Long-read sequencing was performed using Oxford Nanopore Technologies MinION.

Main Results:

• High-purity DNA with an average length of 12-25 kb was consistently obtained.
• Tissue-specific DNA sequencing metrics were generated.
• Mitochondrial DNA consensus sequences were assembled, enabling phylogenetic analysis.
• The methodology provides a framework for processing field specimens for PCR-free sequencing.

Conclusions:

• The described methodology enables efficient, high-quality DNA extraction from millipedes, facilitating genomic studies.
• This approach, utilizing accessible sequencing technology, supports the characterization of genomes in underrepresented arthropod groups.
• The study provides a foundation for future gene-specific analyses and phylogenetic studies in Diplopoda.