Protocol for preparing Drosophila genomic DNA to create chromosome-level de novo genome assemblies

De novogenome assemblies are common tools for examining novel biological phenomena in non-model organisms. Here, we present a protocol for preparingDrosophilagenomic DNA to create chromosome-levelde novogenome assemblies. We describe steps for high-molecular-weight DNA preparation with phenol or Genomic-tips, quality control, long-read nanopore sequencing, short-read DNA library preparation, and sequencing. We then detail procedures of genome assembly, annotation, and assessment that can be used for downstream comparison and functional analysis. For complete details on the use and execution of this protocol, please refer to Sperling etal.1 ; © 2024 The Author(s). Under a Creative Commonslicense - Attribution 4.0 International ; The genomics analysis was supported byNIH/NIDAU01DA047638andNIH/NIGMSR01GM123489to E.G. The biological work was supported by theLeverhulme TrustProject GrantRPG-2018-229and theWellcome TrustInstitutional Strategic Support Fund (RG89305) to D.M.G. and A.L.S. ; A.L.S. designed this study, performed all biological experiments, and wrote the first draft of the original manuscript and this protocol. D.K.F. performed all genome annotations. E.G. performed all genomics-related analyses. A.L.S., D.K.F., E.G., and D.M.G. contributed to choices in methodology and experimental design. Funding for the genomics analysis was obtained by E.G. The funding for all biological experiments was obtained by A.L.S. and D.M.G. ; All code is publicly available. The genome assembly, analysis, and quality control code are onhttps://github.com/ekg/drosophila. The annotation, transcriptomics analysis, and quality control code are onhttps://github.com/FabianDK/Dmerc. All raw and analyzedD.mercatorum genomic data is on the European Nucleotide Archive (ENA): PRJEB64421. The transcriptomics data is on ENA: PRJEB43100. This study did not generate new unique reagents. ; The authors declare no competing interests.