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

Abstract

Obtaining adequate DNA for long-read genome sequencing remains a roadblock to producing contiguous genomes from small-bodied organisms, hindering understanding of phylogenetic relationships and genome evolution. Multiple displacement amplification (MDA) leverages Phi29 DNA polymerase to produce micrograms of DNA from picograms of input. MDA's inherent biases in amplification related to GC and repeat content and chimera production are a problem for long-read genome assembly, which has been little investigated. We explored the utility of MDA for generating template DNA for HiFi sequencing directly from living cells of Caenorhabditis elegans (Nematoda) and Lepidodermella squamata (Gastrotricha) containing one order of magnitude less DNA than required for the PacBio Ultra-Low DNA Input Workflow. HiFi sequencing of libraries prepared from MDA DNA resulted in highly contiguous and complete genomes for both C. elegans (102 Mbp assembly; 336 contigs; N50=868 Kbp; L50=39; BUSCO_nematoda_nucleotide: S:96.1%, D:2.8%) and L. squamata (122 Mbp assembly; 157 contigs; N50=3.9 Mbp; L50=13; BUSCO_metazoa_nucleotide: S:80.8%, D:2.8%). Coverage uniformity for reads from MDA DNA (Gini Index: 0.14, normalized mean across all 100 Kbp blocks: 0.49) and reads from pooled nematode DNA (Gini Index: 0.16, normalized mean across all 100 Kbp blocks: 0.49) proved similar. Using this approach, we sequenced the genome of the microscopic invertebrate Lepidodermella squamata (Gastrotricha), the first of its phylum. Using the newly sequenced genome, we infer Gastrotricha's long-debated phylogenetic position as the sister taxon of Platyhelminthes and conduct a comparative analysis of the Hox cluster.