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179 new drosophilid genomes #Drosophila #Diptera #Drosophilidae #Genome
bioRxiv · Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of LifeLong-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 (>1Mb 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.
### Competing Interest Statement
The authors have declared no competing interest.
@DarrenObbard great to see genome assembly from single fly! Open opportunity for other Dipteran families too.
How do you get the cost down to $150 without pooling individuals? I thought that the cost of an ONT flow cell was higher than that?
Hi! @benoitevol, I'm afraid my main contributions were fly collecting and editing! I think the flowcells are washed, to run multiple libraries sequentially https://www.protocols.io/view/dna-extraction-and-nanopore-library-prep-from-sing-dm6gpbdn8lzp/v2
protocols.io · DNA extraction and Nanopore library prep from single wild-caught drosophilidsWe have been assembling the genomes of many Drosophila species. This protocol fork allows for the sequencing of single wild-caught flies using Nanopore MinION sequencers, in con...