Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment

Aaron Pomerantz, Kristoffer Sahlin, Nina Vasiljevic, Adeline Seah, Marisa Lim, Emily Humble, Susan Kennedy, Henrik Krehenwinkel, Sven Winter, Rob Ogden, Stefan Prost

Published: 2022-04-10 DOI: 10.1038/s41596-022-00682-x

miniaturized laboratory equipment

Supplementary information

An example MinION reads dataset generated using the outlined protocol (3,000 reads). The file contains reads of three fish species: the Atlantic cod ( Gadus morhua ), the Haddock ( Melanogrammus aeglefinus ) and the Whiting ( Merlangius merlangus ), sequenced on a Flongle flow cell. This is previously unpublished data.

Supplementary Data 2

An example index file for demultiplexing with minibar (can be used to demultiplex the example read data in Supplementary Data 1).

Supplementary Data 3

An example file of the primer sequences in fasta format (can be used for primer removal using NGSpeciesID).

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Supplementary information

Untergasser, A. et al. Primer3—new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).
Pomerantz, A. et al. Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building. Gigascience 7, giy033 (2018).
Singh, V. & Kumar, A. PCR primer design. Mol. Biol. Today 2, 27–32 (2001).
Dirzo, R. et al. Defaunation in the Anthropocene. Science 345, 401–406 (2014).
Barnosky, A. D. et al. Approaching a state shift in Earth’s biosphere. Nature 486, 52–58 (2012).
Diagne, C. et al. High and rising economic costs of biological invasions worldwide. Nature 592, 571–576 (2021).
Seebens, H. et al. Global rise in emerging alien species results from increased accessibility of new source pools. Proc. Natl Acad. Sci. USA 115, E2264–E2273 (2018).
Hebert, P. D. N., Ratnasingham, S. & de Waard, J. R. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270, S96–S99 (2003).
Ratnasingham, S. & Hebert, P. bold: The Barcode of Life Data System. Mol. Ecol. Notes 7, 355–364 (2007).
Mizrachi, I. Chapter 1: GenBank: The Nucleotide Sequence Database (NCBI, 2013); https://www.ncbi.nlm.nih.gov/books/NBK470040/
Shokralla, S. et al. Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform. Sci. Rep. 5, 9687 (2015).
Martinez, B. et al. Technology innovation: advancing capacities for the early detection of and rapid response to invasive species. Biol. Invasions 22, 75–100 (2020).
Krehenwinkel, H., Pomerantz, A. & Prost, S. Genetic biomonitoring and biodiversity assessment using portable sequencing technologies: current uses and future directions. Genes 10, 858 (2019).
Menegon, M. et al. On site DNA barcoding by nanopore sequencing. PLoS ONE 12, e0184741 (2017).
Blanco, M. B. et al. Next-generation technologies applied to age-old challenges in Madagascar. Conserv. Genet. 21, 785–793 (2020).
Chang, J. J. M., Ip, Y. C. A., Ng, C. S. L. & Huang, D. Takeaways from mobile DNA barcoding with BentoLab and MinION. Genes 11, 1121 (2020).
Johnson, S. S., Zaikova, E., Goerlitz, D. S., Bai, Y. & Tighe, S. W. Real-time DNA sequencing in the Antarctic Dry Valleys using the Oxford Nanopore Sequencer. J. Biomol. Tech. 28, 2–7 (2017).
Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 530, 228–232 (2016).
Faria, N. R. et al. Mobile real-time surveillance of Zika virus in Brazil. Genome Med. 8, 97 (2016).
Watsa, M., Erkenswick, G. A., Pomerantz, A. & Prost, S. Portable sequencing as a teaching tool in conservation and biodiversity research. PLoS Biol. 18, e3000667 (2020).
Salazar, A. N. et al. An educational guide for nanopore sequencing in the classroom. PLoS Comput. Biol. 16, e1007314 (2020).
Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239 (2016).
Weirather, J. L. et al. Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis. F1000Research 6, 1–32 (2017).
Wick, R. R., Judd, L. M. & Holt, K. E. Deepbinner: demultiplexing barcoded Oxford Nanopore reads with deep convolutional neural networks. PLoS Comput. Biol. 14, e1006583 (2018).
Krehenwinkel, H. et al. Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale. Gigascience 8, giz006 (2019).
Srivathsan, A. et al. Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing. BMC Biol. 17, 96 (2019).
Vasiljevic, N. et al. Developmental validation of Oxford Nanopore Technology MinION sequence data and the NGSpeciesID bioinformatic pipeline for forensic genetic species identification. Forensic Sci. Int. Genet. 53, 102493 (2021).
Maestri, S. et al. A rapid and accurate MinION-based workflow for tracking species biodiversity in the field. Genes 10, 468 (2019).
Seah, A., Lim, M. C. W., McAloose, D., Prost, S. & Seimon, T. A. MinION-based DNA barcoding of preserved and non-invasively collected wildlife samples. Genes 11, 445 (2020).
Srivathsan, A. et al. MinION barcodes: biodiversity discovery and identification by everyone, for everyone. Preprint at BioRxiv https://doi.org/10.1101/2021.03.09.434692 (2021).
Atkins, P. A. P., Gamo, M. E. S. & Voytas, D. F. Analyzing plant gene targeting outcomes and conversion tracts with nanopore sequencing. Int. J. Mol. Sci. 22, 9723 (2021).
Simmons, D. R. et al. The Collection of Zoosporic Eufungi at the University of Michigan (CZEUM): introducing a new repository of barcoded Chytridiomyceta and Blastocladiomycota cultures. IMA Fungus 11, 20 (2020).
Sahlin, K., Lim, M. C. W. & Prost, S. NGSpeciesID: DNA barcode and amplicon consensus generation from long-read sequencing data. Ecol. Evol. 11, 1392–1398 (2021).
Taberlet, P. et al. Power and limitations of the chloroplast trn L (UAA) intron for plant DNA barcoding. Nucleic Acids Res. 35, e14 (2007).
Dieffenbach, C. W., Lowe, T. M. & Dveksler, G. S. General concepts for PCR primer design. Genome Res. 3, S30–S37 (1993).
Ye, J. et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 13, 134 (2012).
Bohmann, K. et al. Strategies for sample labelling and library preparation in DNA metabarcoding studies. Mol. Ecol. Res. 00, 1–16 (2021).
Smyth, R. P. et al. Reducing chimera formation during PCR amplification to ensure accurate genotyping. Gene 469, 45–51 (2010).
Zizka, V. M. A., Elbrecht, V., Macher, J.-N. & Leese, F. Assessing the influence of sample tagging and library preparation on DNA metabarcoding. Mol. Ecol. Resour. 19, 893–899 (2019).
Arulandhu, A. J. et al. Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples. Gigascience 6, gix080 (2017).
Schnell, I. B., Bohmann, K. & Gilbert, M. T. P. Tag jumps illuminated—reducing sequence-to-sample misidentifications in metabarcoding studies. Mol. Ecol. Resour. 15, 1289–1303 (2015).
Lange, V. et al. Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing. BMC Genomics 15, 63 (2014).
Labrador, K., Agmata, A., Palermo, J. D., Follante, J. & Pante, Ma. J. Authentication of processed Philippine sardine products using Hotshot DNA extraction and minibarcode amplification. Food Control 98, 150–155 (2019).
Truett, G. E. et al. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and Tris (HotSHOT). BioTechniques 29, 52–54 (2000).
Knot, I. E., Zouganelis, G. D., Weedall, G. D., Wich, S. A. & Rae, R. DNA barcoding of nematodes using the MinION. Front. Ecol. Evol. https://doi.org/10.3389/fevo.2020.00100 (2020).
Wurzbacher, C. et al. Introducing ribosomal tandem repeat barcoding for fungi. Mol. Ecol. Resour. 19, 118–127 (2019).
Wilson, B. D., Eisenstein, M. & Soh, H. T. High-fidelity nanopore sequencing of ultra-short DNA targets. Anal. Chem. 91, 6783–6789 (2019).
Cornelis, S., Gansemans, Y., Deleye, L., Deforce, D. & Van Nieuwerburgh, F. Forensic SNP Genotyping using Nanopore MinION Sequencing. Sci. Rep. 7, 41759 (2017).
Srivathsan, A. et al. A MinIONTM-based pipeline for fast and cost-effective DNA barcoding. Mol. Ecol. Resour. 18, 1035–1049 (2018).
Sahlin, K. & Medvedev, P. in Research in Computational Molecular Biology (ed. Cowen, L. J.) 227–242 (Springer, 2019).
Vaser, R., Sović, I., Nagarajan, N. & Šikić, M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 27, 737–746 (2017).
Daily, J. Parasail: SIMD C library for global, semi-global, and local pairwise sequence alignments. BMC Bioinform. 17, 81 (2016).
Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).
Byagathvalli, G., Pomerantz, A., Sinha, S., Standeven, J. & Bhamla, M. S. A 3D-printed hand-powered centrifuge for molecular biology. PLoS Biol. 17, e3000251 (2019).
Madden, T. Appendices. BLAST Command Line Applications User Manual [Internet] (Bethesda (MD): National Center for Biotechnology Information (US, 2021); https://www.ncbi.nlm.nih.gov/books/NBK279684/
Watsa, M., Wildlife Disease Surveillance Focus Group. Rigorous wildlife disease surveillance. Science 369, 145–147 (2020).

Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment

Supplementary information

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