One fly-one genome: chromosome-scale genome assembly of a single outbred Drosophila melanogaster

Abstract

A high quality genome assembly is a vital first step for the study of an organism. Recent advances in technology have made the creation of high quality chromosome scale assemblies feasible and low cost. However, the amount of input DNA needed for an assembly project can be a limiting factor for small organisms or precious samples. Here we demonstrate the feasibility of creating a chromosome scale assembly using a hybrid method for a low input sample, a single outbred Drosophila melanogaster. Our approach combines an Illumina shotgun library, Oxford nanopore long reads, and chromosome conformation capture for long range scaffolding. This single fly genome assembly has a N50 of 26 Mb, a length that encompasses entire chromosome arms, contains 95% of expected single copy orthologs, and a nearly complete assembly of this individual's Wolbachia endosymbiont. The methods described here enable the accurate and complete assembly of genomes from small, field collected organisms as well as precious clinical samples.

Figure 1.

Experimental flow chart. A heterozygous fly (H3) was produced by crossing ISO1 and I38 strains. A single female offspring was laterally dissected. From the posterior half, HMW DNA was extracted and used to prepare the two primary assemblies, a R2C2 genomic library for nanopore sequencing, and a Tn5 tagmentation library for paired end Illumina sequencing. The anterior portion was used to isolate intact chromatin to generate a Hi-C paired end Illumina library. The two primary assemblies were merged into one then arranged into chromosome length scaffolds using the Hi-C contact frequency data.

Figure 2.

Genome Contiguity. (A) The read density map for Hi-C read pairs mapped onto the five largest contigs in our final assembly. (B) Dot plot of Hi-C scaffold assembly mapped to the dm6 reference genome. Continuous diagonal lines represent full length scaffolds of all major chromosome arms. For clarity of visualization, we restricted this plot to alignments of 5 kb or more using delta-filter in the mummerplot package.

Figure 3.

Dot-plot comparison of our nearly-complete Wolbachia assembly to the canonical wMel Wolbachia genome sequence. Note that the apparent discontinuity in the top right/left, reflects the circular nature of the bacterial genome, and simply indicates that our assembly breaks the circle at a slightly different place.