Tissue-specific DNA isolation from dissected millipedes for nanopore sequencing

There are approximately 12,000 described species within the class Diplopoda. Only five species, falling within 4 of 16 described orders, have fully sequenced genomes. No whole genomes are available for incredibly diverse families like Xystodesmidae. Furthermore, genetic information attributed to key functions in these species is very limited. There is a growing interest in characterizing genomes of non-model organisms, however, extracting high-quality DNA for organisms with complex morphology can be challenging. Here we describe a detailed methodology for obtaining high-purity DNA from legs, head, and body tissues from wild-caught specimens of the millipede species <i>Cherokia georgiana</i>. Our dissection protocol separates the digestive tract minimizing microbial abundance in the extracted DNA sample. We describe sample homogenization steps that improve total DNA yield. To assess sample quality, concentration, and size we use spectrophotometry, fluorometry, and automated electrophoresis, respectively. We consistently obtain average DNA length upwards of 12-25 kb. We applied Oxford Nanopore Technologies MinION long-read sequencing, an affordable and accessible option with potential for field-based applications. Here we present tissue-specific DNA sequencing metrics, alignment and assembly of mitochondrial DNA consensus sequence, and phylogenetic analysis. While noting the limitations of our nanopore-based sequencing methodology, we provide a framework to process field specimens for PCR-free DNA sequencing data that can be used for gene-specific alignment and analysis.