Comparing the accuracy and efficiency of third generation DNA barcode sequencing: Oxford Nanopore Technologies versus Pacific Biosciences

At times of drastic decrease in biodiversity and loss of species, sometimes referred to as the “sixth mass extinction” or “Holocene extinction”, there is a high demand on the development of effective tools for studying and monitoring biodiversity. In the past decade, new promising technologies, such as third generation sequencing (TGS), enabled massive, rapid, and cost-effective data analysis of non-model organisms, accelerating taxonomic identification studies and contributing to conservation applications. Here, we focus on the comparison of the two main TGS providers, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT), for the purpose of DNA barcoding. For ONT, we also tested selected combinations of different types of flow cells and ligation sequencing kits. Out of five tested combinations (PacBio, ONT Flongle flow cell & SQK-LSK110 kit, R9 flow cell & SQK-LSK109 kit, R9 & SQK-LSK100 kit, and R10 flow cell & Q20+ chemistry kit), ONT’s Flongle turned out to be most variable in returning the results, but at the same time the most cost efficient. The highest numbers of successfully sequenced samples were achieved with the ONT’s R10 & Q20+ chemistry combination. In terms of library preparation time, ONT protocols are the quickest, whereas regarding cost effectiveness - using Sanger pricing per sample as a cut-off - various technologies become affordable depending on the number of samples used. Although both tested platforms are suitable for DNA barcoding, we further discuss their limitations and applicability to different studies, with a special focus on the price and the number of samples. The pipeline we developed, from whole specimens to final DNA barcode consensuses, can aid planning and budgeting biodiversity studies, maximising the number of specimens sequenced in one run and speeding up the sample processing time. ### Competing Interest Statement The authors have declared no competing interest.