Incidence and attributes of chimeric COI and 18S rDNA sequences derived from nematode-infected arthropods

PCR amplification of mixed-template samples can generate chimeric sequences, molecules that combine partial sequences from two or more source organisms. These artefacts pose a significant challenge for high-throughput sequencing studies, generating both type I errors (true species mistakenly rejected) and type II errors (chimeras incorrectly viewed as valid species). Despite their impact, the incidence, structural properties, and factors driving chimera formation remain poorly understood.

Using nanopore sequencing of 531 nematode-infected arthropod specimens, chimera formation was characterized in two widely used gene regions: the 658 bp of cytochrome c oxidase subunit I (COI) barcode and a 900 bp segment of 18S ribosomal DNA (18S rDNA). Chimeras were detected for both gene regions despite the deep sequence divergences between members of these two phyla. However, the incidence of chimeric molecules was higher for 18S rDNA than COI with chimera formation correlating with local variation in DNA stability and conserved sequence tracts between parent species.

By clarifying the associations between DNA stability, sequence conservation, and chimera formation, this study offers insights that can inform the development of more robust approaches to chimera detection.