Tissue specific millipede DNA extraction and mitochondrial DNA analysis with Nanopore sequencing

There are approximately 13,000 described species within the class Diplopoda. Only five species, falling within four of sixteen 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 gDNA from legs, head, and body tissues from wild-caught specimens of the millipede species Cherokia georgiana. Our dissection protocol separates the digestive tract minimizing microbial abundance in the extracted DNA sample. We describe sample homogenization steps that improve total gDNA yield. To assess sample quality, concentration, and size we use spectrophotometry, fluorometry, and automated electrophoresis, respectively. We obtain gDNA length ranging between 12-25 kb. In a preliminary sequencing feasibility test using head tissue gDNA, we apply Oxford Nanopore Technologies (ONT) MinION long-read sequencing obtaining an N50 score of 4.75 kb. Generated FAST5 files were aligned to a reference using Minimap2 within the Geneious Prime software platform for further phylogenetic analysis.There are approximately 13,000 described species within the class Diplopoda. Only five species, falling within four of sixteen 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 gDNA from legs, head, and body tissues from wild-caught specimens of the millipede species Cherokia georgiana. Our dissection protocol separates the digestive tract minimizing microbial abundance in the extracted DNA sample. We describe sample homogenization steps that improve total gDNA yield. To assess sample quality, concentration, and size we use spectrophotometry, fluorometry, and automated electrophoresis, respectively. We obtain gDNA length ranging between 12-25 kb. In a preliminary sequencing feasibility test using head tissue gDNA, we apply Oxford Nanopore Technologies (ONT) MinION long-read sequencing. Here we provide the raw sequencing data collected as part of this project.