Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Baylor College of Medicine Houston Texas United States of America

Center for Reproductive Evolution Department of Biology Syracuse University Syracuse New York United States of America

CZ Biohub Investigator San Francisco California United States of America

Daintree Rainforest Observatory James Cook University Townsville Australia

Department of Biological and Environmental Science University of Jyväskylä Jyväskylä Finland

Department of Biological Sciences Hokkaido University Sapporo Japan

Department of Biological Sciences Michigan Technological University Houghton Michigan United States of America

Department of Biological Sciences Tokyo Metropolitan University Tokyo Japan

Department of Biological Sciences Virginia Tech Blacksburg Virginia United States of America

Department of Biology Case Western Reserve University Cleveland Ohio United States of America

Department of Biology Stanford University Stanford California United States of America

Department of Biology University of North Carolina Chapel Hill Chapel Hill North Carolina United States of America

Department of Cell and Molecular Biology University of California Berkeley Berkeley California United States of America

Department of Complexity Science and Engineering The University of Tokyo Tokyo Japan

Department of Developmental Biology Stanford University Stanford California United States of America

Department of Ecology and Evolutionary Biology Yale University New Haven Connecticut United States of America

Department of Entomology Cornell University Ithaca New York United States of America

Department of Evolution and Ecology University of California Davis Davis California United States of America

Department of Zoology The University of British Columbia Vancouver Canada

Division of Genetic Medicine Department of Pediatrics; Department of Laboratory Medicine and Pathology University of Washington Seattle Washington United States of America

Hawaii Invertebrate Program Division of Forestry and Wildlife Honolulu Hawaii United States of America

Hokkaido University Museum Hokkaido University Sapporo Japan

Howard Hughes Medical Institute University of California Berkeley Berkeley California United States of America

Institute of Ecology and Evolution University of Edinburgh Edinburgh United Kingdom

Institute of Entomology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Pacific Biosciences Research Center University of Hawai'i Mānoa Hawaii United States of America

School of Environmental and Natural Sciences Bangor University Bangor United Kingdom

School of Life Sciences University of Nevada Las Vegas Las Vegas Nevada United States of America

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bioRxiv. 2023 Oct 02:2023.10.02.560517. doi: 10.1101/2023.10.02.560517. PubMed

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