On-site sequencing

Recent advances in molecular biology allow the use of genetic markers (DNA barcodes) to support and confirm morphological evidence for species identification and to quantify interspecific differences in order to compare species in terms of evolutionary distance. Most barcodes are still generated using the Sanger sequencing method, which requires access to a well-equipped molecular biology laboratory. Second-generation sequencing technologies are also used for barcoding, but they depend on expensive equipment and the reads are often too short to distinguish species reliably. We have demonstrated that the third-generation sequencer Oxford Nanopore Technologies (ONT) can successfully sequence under extreme field conditions such as the tropical rainforests of Tanzania, Congo and Borneo. We keep implementing new workflows for barcoding in the field, from DNA extraction to the generation of consensus sequences. The selected protocols allow the extraction of DNA from tiny snail-tissue biopsies and from whole beetles after cutting the abdomen to release soft tissues, as required to preserve the integrity of the specimens for detailed morphological evaluation. PCR products are successfully obtained despite the transport of our equipment in a standard Peli case and the storage of DNA at room temperature and of molecular biology reagents in local fridges and freezers powered for only 10 h per day. The MinION flow cells, which are not adversely affected by the transportation and storage conditions, retained most of their active pores and produce a good number of reads in a few hours. On the software side, the new bioinformatics pipeline allow us to analyze MinION reads using open-source and custom-developed scripts that run locally on a Linux Virtual Machine. The sequencing and data analysis can therefore be combined on a standard Windows laptop with a user-friendly interface.

Visit the Multimedia page for on-site sequencing videos!

Tanzania, May 2015
Borneo, Malaysia, April 2016
Congo, February 2017

Borneo, Malaysia, September 2017
Borneo, Malaysia, March 2018
Durmitor, Montenegro July 2018
Borneo, Brunei, September 2018
Durmitor, Montenegro July 2019
Borneo, Brunei, September 2019
Tuscany, Italy, May 2022
Durmitor, Montenegro July 2023
Borneo, Brunei, September 2023

Gobi desert, Mongolia, 2022
Phylippines, November 2022
Mizoram, India, March 2023

Tanganika lake, Congo expedition (2016)

“Twenty years from now you will be more disappointed by the things you didn’t do than by the ones you did do. So throw off the bowlines! Sail away from safe harbor. Catch the trade winds in your sails. Explore. Dream. Discover!”
..Mark Twain (unverified)

Publications related to on-site sequencing

Menegon M. et al (2017). On site DNA barcoding by nanopore sequencing. PLoS One 12(10):e0184741.
Maestri S. et al (2019). A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field. Genes Jun 20;10(6)
Schilthuizen M. et al (2019) Bringing the lab to the field: a new lowland Microparmarion semi-slug (Gastropoda: Ariophantidae) described and DNA-barcoded in the forest. J. Molluscan Studies 85: 35-40
Freitag Het al (2021) Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along with further records of Hydraena spp. from Durmitor National Park, Montenegro and comments on the DNA barcoding problem with the genus. Biodivers Data J. 2021 Jan 12;9:e59892.
Marcolungo L. et al (2022) Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing. Pathogens doi: 10.3390/pathogens11020199.
Schilthuizen M, Thompson CG, de Vries R, van Peursen ADP, Paterno M, Maestri S, Marcolongo L, Esposti CD, Delledonne M, Njunjić I (2022) A new giant keelback slug of the genus Limax from the Balkans, described by citizen scientists. Biodiversity Data Journal 10: e69685. https://doi.org/10.3897/BDJ.10.e69685
Schilthuizen M, Berenyi S, Ezzwan NSMN, Hamdani NIAA, Wu H, De Antoni L, Vincenzi L, de Gier W, van Peursen ADP, Njunjić I, Delledonne M, Slik F, Grafe U, Cicuzza D (2023) A new semi-slug of the genus Microparmarion from Brunei, discovered, described and DNA-barcoded on citizen-science 'taxon expeditions' (Gastropoda, Stylommatophora, Ariophantidae). Biodiversity Data Journal 11: e101579. https://doi.org/10.3897/BDJ.11.e101579
Otani S, Bertoli L, Lucchini F, van den Beuken TPG, Boin D, Ellis L, Friedrich H, Jacquot B, Kountouras S, Lim SR, Nigro E, Su’eif S’ie, Tan WH, Grafe U, Cicuzza D, Delledonne M, Njunjić I, Schilthuizen M (2024) A new, unusually large, Clavicornaltica Scherer, 1974 flea beetle from Borneo, described and sequenced in the field by citizen scientists (Coleoptera, Chrysomelidae, Galerucinae). Biodiversity Data Journal 12: e119481. https://doi.org/10.3897/BDJ.12.e119481

On-site sequencing

“Twenty years from now you will be more disappointed by the things you didn’t do than by the ones you did do. So throw off the bowlines! Sail away from safe harbor. Catch the trade winds in your sails. Explore. Dream. Discover!”..Mark Twain (unverified)

Publications related to on-site sequencing

On-site
sequencing

“Twenty years from now you will be more disappointed by the things you didn’t do than by the ones you did do. So throw off the bowlines! Sail away from safe harbor. Catch the trade winds in your sails. Explore. Dream. Discover!”
..Mark Twain (unverified)