A flexible low-hazard protocol for sample preparation and high-molecular weight DNA extraction from large lepidopterans

Large insects are an optimal system for genomics studies given the ease with which large quantities of genetic material can be obtained from single individuals. This avoids the need to generate inbred/clonal genotypes or complications associated with sequencing pooled samples of heterogenous individuals. The advent of third generation long-read sequencing has made it easier than ever to produce high quality genome assemblies for non-model species, but acquiring suitable high-molecular weight (HMW) DNA can be a difficult process that often requires the use of hazardous chemicals such as phenol or cetyltrimethylammonium bromide (CTAB). Here, we describe a protocol for extracting HMW DNA from large insects (specifically Manduca sexta) with minimal use of hazardous materials. This protocol is suitable for use on small insects, but larger insects must be dissected prior to DNA extraction. As such, we have also included a detailed protocol for preparing tissues of either adult or larval samples.