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Building the Adriatic Calanoid Database with the help of Oxford’s MinION system

Metabarcoding seems to be the future of biodiversity research. Simplifying time-consuming taxonomical methods, molecular approaches attract more and more ecology labs to join the new way of ecosystem evaluation each year. To achieve creditable results, reliable reference databases are needed. In response to this need we started building our own Adriatic multi-marker genetic database of calanoid copepods for our ongoing project. In this study we wanted to test the capabilities of the MinION sequencer for our purposes. All our samples were taken from our South Adriatic and North Adriatic coastal sampling stations through 2021. For the test, we used 42 specimens of calanoids with 4 markers (COI, 18S, 28S, ITS1-5.8S- ITS2) with fragment lengths ranging between 900-1300 BPs. We used previously acquired Sanger sequencing data to compare and verify our results. In the sequencing run 92.5% of the sequences were recovered with 99.95% final accuracy. 48% of the errors turned out to be indels with 1:2 deletion, insertion ratio. The most common errors were related to cytosine and guanine bases. The comparison of the two datasets also revealed, that sites with nucleotide repeats can cause ambiguous bases in the Sanger final product, while Nanopore resolved these sites at ease. The only drawback of the method seems to be its sensitivity for alien contamination, while using universal primers. In conclusion: with appropriate pre-and post-processing, MinION sequencing, with its ability to generate long barcodes is a viable alternative for Sanger sequencing in building genetic databases.

barcoding database ; nanopore ; calanoid copepods ; Adriatic Sea

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