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

Area of Science:

• Genomics
• Evolutionary Biology
• Bioinformatics

Background:

• Limited genetic material is a major challenge in biodiversity and genome evolution studies.
• Existing methods struggle with small sample sizes for comprehensive genomic analysis.

Purpose of the Study:

• To develop and validate a robust method for amplifying and sequencing genomic DNA and mRNA from limited nematode samples.
• To investigate genome size, gene prediction accuracy, and phylogenetic relationships in early-branching nematode species.

Main Methods:

• Multiple Displacement Amplification (MDA) for genomic DNA and Smartseq2 for mRNA amplification.
• Long-read sequencing using Oxford Nanopore Technologies (ONT).
• Genome assembly, transcriptome sequencing, and phylogenomic analyses.

Main Results:

• Achieved 98% genome coverage from nanograms of DNA, despite reduced coverage in repetitive regions.
• Demonstrated improved accuracy and completeness of gene predictions using combined assembly and transcriptome data compared to de novo approaches.
• Identified larger genome sizes in basal Chromadoria and Enoplia species.
• Fully assembled nine mitogenomes and resolved strong phylogenetic support for Enoplia as sister to the rest of Nematoda.

Conclusions:

• MDA combined with ONT sequencing is a robust method for generating high-quality genomic data from limited biological samples.
• This approach significantly advances the study of genome diversity and evolution within the phylum Nematoda.
• The findings provide new insights into nematode phylogeny and genome characteristics.