A proof-of-principle study: Species identification based on mitochondrial 12S rRNA gene using QNome nanopore...

Accurate species identification can provide crucial evidence to aid criminal investigations and preserve species diversity. DNA barcoding is an effective tool to ascertain the origin of degraded, trace, or processed samples, and the mitochondrial 12S rRNA gene serving as a potential marker due to its low intraspecific variation and high interspecific variation. QNome nanopore sequencing, with its flexible read lengths, real-time analysis, and portable device size, offers new means for species identification. In this proof-of-principle study, we collected 12S rRNA gene reference sequences of 120 common species from Genbank genetic sequence database and conducted sequence similarity analysis to evaluate the applicability of this genetic marker. The automated species identification pipeline, ClassIdent, based on QNome nanopore sequencing of the 12S rRNA gene, was subsequently developed as an open-source tool. We sequenced 60 tested samples from different animals using the QNome sequencer, and ClassIdent was employed to detect matching proportions and generate consensus sequences. Out of the 60 tested samples, 53 matched the sequences of their corresponding species, while the remaining seven matched sequences from closely related species within the same genus or family. The average proportion of sequencing reads assigned to the reference sequence was 95.66 %, with an average sequence similarity of 99.11 %. The consensus sequences generated by ClassIdent demonstrated high accuracy, showing an average sequence similarity of 99.34 % compared to Sanger sequences. Overall, this pipeline demonstrated the potential of using 12S rRNA gene for species identification via Qnome nanopore sequencing. However, further research is needed to incorporate more DNA barcoding markers or to detect the whole mitochondrial genome based on long read sequencing technology.