Single-worm long-read sequencing reveals genome diversity in free-living nematodes

Abstract

Obtaining sufficient genetic material from a limited biological source is currently the primary operational bottleneck in studies investigating biodiversity and genome evolution. In this study, we employed multiple displacement amplification (MDA) and Smartseq2 to amplify nanograms of genomic DNA and mRNA, respectively, from individual Caenorhabditis elegans. Although reduced genome coverage was observed in repetitive regions, we produced assemblies covering 98% of the reference genome using long-read sequences generated with Oxford Nanopore Technologies (ONT). Annotation with the sequenced transcriptome coupled with the available assembly revealed that gene predictions were more accurate, complete and contained far fewer false positives than de novo transcriptome assembly approaches. We sampled and sequenced the genomes and transcriptomes of 13 nematodes from early-branching species in Chromadoria, Dorylaimia and Enoplia. The basal Chromadoria and Enoplia species had larger genome sizes, ranging from 136.6 to 738.8 Mb, compared with those in the other clades. Nine mitogenomes were fully assembled, and displayed a complete lack of synteny to other species. Phylogenomic analyses based on the new annotations revealed strong support for Enoplia as sister to the rest of Nematoda. Our result demonstrates the robustness of MDA in combination with ONT, paving the way for the study of genome diversity in the phylum Nematoda and beyond.

Revisiting genomes of non-model species with long reads yields new insights into their biology and evolution

“…Considering that TEs represent 289 Mb of the 359-Mb genome, we can estimate that a large aspect of this genome was completely overlooked in the past. A recent study has shown that genome assemblies from basal nematodes contain more repeats (ranging from 23.4% up to 50.6% repeats) than nematodes belonging to other clades (ranging from 0.8 %p to 31%) [53]. The results here presented are consistent with the previousfindings as R. culicivorax , a basal nematode, showed a high repeat and TE content and the derived P. sp.…”

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

“…This would result in fewer free 3’ ends of strands that could anneal to another strand and act as a primer to initiate chimera formation. As chimeric reads may be more problematic in data obtained from more fragmented template DNA, paired-end Illumina data generated from half of the specimen lysate before MDA could be used in future studies to aid in the detection of chimeras (see Lee et al 2023). Examining the mapping of paired-end short reads to post-amplification HiFi reads could be used to reveal chimeric reads as one of the reads in a pair would be expected to map on one side of the chimeric junction while its mate would be expected to map elsewhere.…”

“…MDA has been explored in the context of Oxford Nanopore (ONT) sequencing to produce relatively contiguous genomes from single nematodes (Lee et al 2023). For C. elegans , the authors generated an assembly consisting of 499 contigs with an N50 of 656.2 Kbp and a BUSCO completeness score of 97.6%, compared to 336 contigs with an N50 of 868.1 Kbp and a BUSCO score of 92.2% in the present study.…”

“…Whole genome amplification (WGA) methodologies are of great use to the fields of diagnostic medicine and single cell research (Evrony et al 2015; Fu et al 2015; Li et al 2018; Zhou et al 2020), but their utility in amplifying DNA for sequencing very small-bodied organisms in the age of long-read sequencing technologies has received little attention (but see Lee et al 2023). WGA is a popular strategy for generating large amounts of DNA from limiting sample material through techniques such as multiple displacement amplification (MDA) (Dean et al 2001), degenerate-oligonucleotide-primed-PCR (DOP-PCR) (Telenius et al 1992), DOP-PCR (Telenius et al 1992), PEP-PCR (Moghaddaszadeh-Ahrabi et al 2012), LM-PCR (Carey MF et al 2009), T-PCR (Tarr AW et al 2005), and multiple annealing and looping based amplification cycles (MALBAC) (Chapman et al 2015).…”

Phylogenomic Insights into the Evolution and Origin of Nematoda

“…several micrograms), typically thousands of times more than that which is contained within a single microscopic individual. Zhang et al (2019) proposed WGA in genome sequencing and phylogenomic of small animals, and two pioneering studies have used this method based on Illumina and PacBio platform to generate nematodes genomes (Lee et al, 2023;Doyle et al, 2019), although no study had yet evaluated its performance using microscopic species. In this study, we demonstrated that WGA generated smaller assembly and more fragmented contigs, probably due to the bias in the amplification process (Sabina et al, 2015).…”