Multiple Displacement Amplification Facilitates SMRT Sequencing of Microscopic Animals and the Genome of the GastrotrichLepidodermella squamata(Dujardin, 1841)

Abstract

Background:Obtaining adequate DNA for long-read genome sequencing remains a roadblock to producing contiguous genomes from small-bodied organisms. Multiple displacement amplification (MDA) leverages Phi29 DNA polymerase to produce micrograms of DNA from picograms of input. Few genomes have been generated using this approach, due to concerns over biases in amplification related to GC and repeat content and chimera production. Here, we explored the utility of MDA for generating template DNA for PacBio HiFi sequencing usingCaenorhabditis elegans(Nematoda) andLepidodermella squamata(Gastrotricha).Results:HiFi sequencing of libraries prepared from MDA DNA produced highly contiguous and complete genomes for bothC. elegans(102 Mbp assembly; 336 contigs; N50 = 868 Kbp; L50 = 39; BUSCO_nematoda: S:92.2%, D:2.7%) andL. squamata(122 Mbp assembly; 157 contigs; N50 = 3.9 Mb; L50 = 13; BUSCO_metazoa: S: 78.0%, D: 2.8%). AmplifiedC. elegansreads mapped to the reference genome with a rate of 99.92% and coverage of 99.75% with just one read (of 708,811) inferred to be chimeric. Coverage uniformity was nearly identical for reads from MDA DNA and reads from pooled worm DNA when mapped to the reference genome. The genome ofLepidodermella squamata, the first of its phylum, was leveraged to infer the phylogenetic position of Gastrotricha, which has long been debated, as the sister taxon of Platyhelminthes.Conclusions:This methodology will help generate contiguous genomes of microscopic taxa whose body size precludes standard long-read sequencing.L. squamatais an emerging model in evolutionary developmental biology and this genome will facilitate further work on this species.