A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species-specific detection in environmental DNA.
droplet digital polymerase chain reaction
metabarcoding marine biosecurity
quantitative PCR
Journal
Molecular ecology resources
ISSN: 1755-0998
Titre abrégé: Mol Ecol Resour
Pays: England
ID NLM: 101465604
Informations de publication
Date de publication:
Nov 2019
Nov 2019
Historique:
received:
18
01
2019
revised:
07
06
2019
accepted:
10
06
2019
pubmed:
12
7
2019
medline:
13
3
2020
entrez:
12
7
2019
Statut:
ppublish
Résumé
Targeted species-specific and community-wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R
Identifiants
pubmed: 31293089
doi: 10.1111/1755-0998.13055
doi:
Substances chimiques
DNA, Environmental
0
RNA, Ribosomal, 18S
0
Types de publication
Comparative Study
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1407-1419Subventions
Organisme : New Zealand Government's Strategic Science Investment Fund (SSIF) through the NIWA Coasts and Oceans Research Programme 6, Marine Biosecurity
ID : SCI 2016-18
Informations de copyright
© 2019 John Wiley & Sons Ltd.
Références
Ammon, U. V., Wood, S. A., Laroche, O., Zaiko, A., Tait, L., Lavery, S., … Pochon, X. (2018). Combining morpho-taxonomy and metabarcoding enhances the detection of non-indigenous marine pests in biofouling communities. Scientific Reports, 8, 16290. https://doi.org/10.1038/s41598-018-34541-1
Andruszkiewicz, E. A., Sassoubre, L. M., & Boehm, A. B. (2017). Persistence of marine fish environmental DNA and the influence of sunlight. PLoS ONE, 12, e0185043. https://doi.org/10.1371/journal.pone.0185043
Aronesty, E. (2011). ea-utils: Command-line tools for processing biological sequencing data. Retrieved from https://github.com/ExpressionAnalysis/ea-utils
Aylagas, E., Borja, Á., Irigoien, X., & Rodríguez-Ezpeleta, N. (2016). Benchmarking DNA Metabarcoding for Biodiversity-Based Monitoring and Assessment. Frontiers in Marine Science, 10. https://www.frontiersin.org/articles/10.3389/fmars.2016.00096/full. https://doi.org/10.3389/fmars.2016.00096
Bhat, S., Curach, N., Mostyn, T., Bains, G. S., Griffiths, K. R., & Emslie, K. R. (2010). Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units. Analytical Chemistry, 82, 7185-7192. https://doi.org/10.1021/ac100845m
Bista, I., Carvalho, G. R., Walsh, K., Seymour, M., Hajibabaei, M., Lallias, D., … Creer, S. (2017). Annual time-series analysis of aqueous eDNA reveals ecologically relevant dynamics of lake ecosystem biodiversity. Nature Communications, 8, 14087. https://doi.org/10.1038/ncomms14087
Borrell, Y. J., Miralles, L., Do Huu, H., Mohammed-Geba, K., & Garcia-Vazquez, E. (2017). DNA in a bottle-Rapid metabarcoding survey for early alerts of invasive species in ports. PLoS ONE, 12, e0183347. 0.1371/journal.pone.0183347
Brown, E. A., Chain, F. J. J., Zhan, A., MacIsaac, H. J., & Cristescu, M. E. (2016). Early detection of aquatic invaders using metabarcoding reveals a high number of non-indigenous species in Canadian ports. Diversity and Distributions, 22, 1045-1059. https://doi.org/10.1111/ddi.12465
Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., … Wittwer, C. T. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611-622. https://doi.org/10.1373/clinchem.2008.112797
Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., … Knight, R. (2010). QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7, 335-336. https://doi.org/10.1038/nmeth.f.303
Chambert, T., Pilliod, D. S., Goldberg, C. S., Doi, H., & Takaharam, T. (2018). An analytical framework for estimating aquatic species density from environmental DNA. Ecology and Evolution, 25, 3468-3477. https://doi.org/10.1002/ece3.3764
Clarke, L. J., Soubrier, J., Weyrich, L. S., & Cooper, A. (2014). Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias. Molecular Ecology Resources, 14, 1160-1170. https://doi.org/10.1111/1755-0998.12265
Cowart, D. A., Pinheiro, M., Mouchel, O., Maguer, M., Grall, J., Miné, J., & Arnaud-Haond, S. (2015). Metabarcoding is powerful yet still blind: A comp, arative analysis of morphological and molecular surveys of seagrass communities. PLoS ONE, 10, e0117562. https://doi.org/10.1371/journal.pone.0117562
Cox, M. P., Peterson, D. A., & Biggs, P. J. (2010). SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinformatics, 11, 485. https://doi.org/10.1186/1471-2105-11-485
Cristescu, M. E., & Hebert, P. D. N. (2018). Uses and misuses of environmental DNA in biodiversity science and conservation. Annual Review of Ecology, Evolution, and Systematics, 49, 209-230. https://doi.org/10.1146/annurev-ecolsys-110617-062306
De Barba, M., Miquel, C., Boyer, F., Mercier, C., Rioux, D., Coissac, E., & Taberlet, P. (2014). DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: Application to omnivorous diet. Molecular Ecology Resources, 14, 306-323. https://doi.org/10.1111/1755-0998.12188
de Vargas, C., Audic, S., Henry, N., Decelle, J., Mahe, F., Logares, R., … Velayoudon, D. (2015). Eukaryotic plankton diversity in the sunlit ocean. Science, 348, 1261605. https://doi.org/10.1126/science.1261605
Deagle, B. E., Jarman, S. N., Coissac, E., Pompanon, F., & Taberlet, P. (2014). DNA metabarcoding and the cytochrome c oxidase subunit I marker: Not a perfect match. Biology Letters, 10, 20140562. https://doi.org/10.1098/rsbl.2014.0562
Deiner, K., Bik, H. M., Mächler, E., Seymour, M., Lacoursière-Roussel, A., Altermatt, F., … Bernatchez, L. (2017). Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology, 26, 5872-5895. https://doi.org/10.1111/mec.14350
DeLong, E. R., DeLong, D. M., & Clarke-Pearson, D. L. (1988). Comparing areas under two or more correlated receiver operating characteristic curves: A nonparametric approach. Biometrics, 44, 837-845.
Dingle, T. C., Sedlak, R. H., Cook, L., & Jerome, K. R. (2013). Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clinical Chemistry, 59, 1670-1672. https://doi.org/10.1373/clinchem.2013.211045
Doi, H., Takahara, T., Minamoto, T., Matsuhashi, S., Uchii, K., & Yamanaka, H. (2015). Droplet digital Polymerase Chain Reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species. Environmental Science and Technology, 49, 5601-5608. https://doi.org/10.1021/acs.est.5b00253
Doi, H., Uchii, K., Takahara, T., Matsuhashi, S., Yamanaka, H., & Minamoto, T. (2015). Use of droplet digital PCR for estimation of fish abundance and biomass in environmental DNA surveys. PLoS ONE, 10, e0122763. https://doi.org/10.1371/journal.pone.0122763
Dowle, E., Pochon, X., Banks, J., Shearer, K., & Wood, S. A. (2016). Targeted gene enrichment and high throughput sequencing for environmental biomonitoring. Molecular Ecology Resources, 16, 1240-1254. https://doi.org/10.1111/1755-0998
Edgar, R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics, 26, 2460-2461. https://doi.org/10.1093/bioinformatics/btq461
Evans, N. T., Li, Y., Renshaw, M. A., Olds, B. P., Deiner, K., Turner, C. R., Pfrender, M. E. (2017). Fish community assessment with eDNA metabarcoding: Effects of sampling design and bioinformatic filtering. Canadian Journal of Fisheries and Aquatic Sciences, 74(9), 1362-1374. https://doi.org/10.1139/cjfas-2016-0306
Gillum, J. E., Jimenez, L., White, D. J., Goldstien, S. J., & Gemmell, N. J. (2014). Development and application of a quantitative real-time PCR assay for the globally invasive tunicate Styela clava. Management of Biological Invasions, 5, 133-142.
Goldberg, C. S., Turner, C. R., Deiner, K., Klymus, K. E., Philip, K., Thomsen, F., … Taberlet, P. (2016). Critical consideration for the application of environmental DNA methods to detect aquatic species. Methods in Ecology and Evolution, 7, 1299-1307. https://doi.org/10.1111/2041-210X.12595
Guillera-Arroita, G., Ridout, M. S., & Morgan, B. J. T. (2010). Design of occupancy studies with imperfect detection. Methods in Ecology and Evolution, 1, 131-139. https://doi.org/10.1111/j.2041-210X.2010.00017
Guillou, L., Bachar, D., Audic, S., Bass, D., Berney, C., Bittner, L., … Christen, R. (2013). The Protist Ribosomal Reference database (PR2): A catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Research, 41, D59-604. https://doi.org/10.1093/nar/gks1160
Hale, S. S., & Heltche, J. F. (2008). Signals from the benthos: Development and evaluation of a benthic index for the nearshore Gulf of Maine. Ecological Indicators, 8, 338-350. https://doi.org/10.1016/j.ecolind.2007.04.004
Hänfling, B., Lawson Handley, L., Read, D. S., Hahn, C., Li, J., Nichols, P., … Winfield, I. J. (2016). Environmental DNA metabarcoding of lake fish communities reflects long-term data from established survey methods. Molecular Ecology, 25, 3101-3119. https://doi.org/10.1111/mec.13660
Hatzenbuhler, C., Kelly, J. R., Martinson, J., Okum, S., & Pilgrim, E. (2017). Sensitivity and accuracy of high-throughput metabarcoding methods for early detection of invasive fish species. Scientific Reports, 13, 46393. https://doi.org/10.1038/srep46393
Haugland, R. A., Siefring, S. C., Wymer, L. J., Brenner, K. P., & Dufour, A. P. (2005). Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Research, 39, 559-568. https://doi.org/10.1016/j.watres.2004.11.011
Hindson, B. J., Ness, K. D., Masquelier, D. A., Belgrader, P., Heredia, N. J., Makarewicz, A. J., … Colston, B. W. (2011). High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical Chemistry, 83, 8604-8610. https://doi.org/10.1021/ac202028g
Hines, J. E. (2006). PRESENCE - Software to estimate patch occupancy and related parameters. USGS-PWRC. Retrieved from http://www.mbr-pwrc.usgs.gov/software/presence.html.
Holloway, M. G., & Keough, M. J. (2002). An introduced polychaete affects recruitment and larval abundance of sessile invertebrates. Ecological Application, 12, 1803-1823. https://doi.org/10.1890/1051-0761
Keeley, N., Wood, S. A., & Pochon, X. (2018). Development and validation of a multi-trophic metabarcoding biotic index for benthic organic enrichment biomonitoring. Ecological Indicators, 85, 1044-1057. https://doi.org/10.1016/j.ecolind.2017.11.014
Kelly, R. P., Port, J. A., Yamahara, K. M., & Crowder, L. B. (2014). Using environmental DNA to census marine fishes in a large mesocosm. PLoS ONE, 9, e86175. https://doi.org/10.1371/journal.pone.0086175.
Kim, T. G., Jeong, S. Y., & Cho, K. S. (2014). Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil. Applied Microbiology Biotechnology, 98, 6105-6113. https://doi.org/10.1007/s00253-014-5794-4
Kreader, C. A. (1996). Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Applied and Environmental Microbiology, 62, 1102-1106.
Lacoursière-Roussel, A., Côté, G., Leclerc, V., & Bernatchez, L. (2016). Quantifying relative fish abundance with eDNA: A promising tool for fisheries management. Journal of Applied Ecology, 53, 1148-1157. https://doi.org/10.1111/1365-2664.12598
Laroche, O., Wood, S. A., Tremblay, L. A., Ellis, J. I., Lejzerowicz, F., Pawlowki, J., … Pochon, X. (2016). First evaluation of foraminiferal metabarcoding for monitoring benthic health at an offshore oil drilling site. Marine Environmental Research, 120, 225-235. https://doi.org/10.1016/j.marenvres.2016.08.009
Lee, C. K., Herbold, C. W., Polson, S. W., Wommack, K. E., Williamson, S. J., McDonald, I. R., & Cary, S. C. (2012). Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing. PLoS ONE, 7(9), e44224. https://doi.org/10.1371/journal.pone.0044224
Leray, M., Yang, J. Y., Meyer, C. P., Mills, S. C., Agudelo, N., Ranwez, V., … Machida, R. J. (2013). A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: Application for characterizing coral reef fish gut contents. Frontiers in Zoology, 10, 14. https://doi.org/10.1186/1742-9994-10-34
Lugg, W. H., Griffiths, J., van Rooyen, A. R., Weeks, A. R., & Tingley, R. (2017). Optimal survey designs for environmental DNA sampling. Methods in Ecology and Evolution, 9(4), 1049-1059. https://doi.org/10.1111/2041-210X.12951
Machida, R. J., Leray, M., Ho, S.-L., & Knowlton, N. (2017). Metazoan mitochondrial gene sequence reference datasets for taxonomic assignment of environmental samples. Scientific Data, 4, 1-7. https://doi.org/10.1038/sdata.2017.27
MacKenzie, D. I., Nichols, J. D., Lachman, G. D., Droege, S., Royle, J. A., & Langtimm, C. A. (2002). Estimating site occupancy rates when detection probabilities are less than one. Ecology, 83, 2248-2255. https://doi.org/10.1890/0012-9658(2002)083[2248:ESORWD]2.0.CO;2
Mahé, F., Rognes, T., Quince, C., de Vargas, C., & Dunthorn, M. (2014). Swarm: Robust and fast clustering method for amplicon-based studies. PeerJ, 2, e593. https://doi.org/10.7717/peerj.593
Murtaugh, P. A. (1996). The statistical evaluation of ecological indicators. Ecological Applications, 6, 132-139. https://doi.org/10.2307/2269559
Nathan, L. R., Simmons, M. D., Wegleitner, B., Jerde, C. L., & Mahon, A. (2014). Quantifying environmental DNA signals for aquatic invasive species across multiple detection platforms. Environmental and Science Technology, 48, 12800-12806. https://doi.org/10.1021/es5034052
Nichols, J. D., Bailey, L. L., O’Connell, A. F. Jr, Talancy, N. W., Campbell Grant, E. H., Gilbert, A. T., … Hines, J. E. (2008). Multi-scale occupancy estimation and modeling using multiple detection methods. Journal of Applied Ecology, 45, 1321-1329. https://doi.org/10.1111/j.1365-2664.2008.01509.x
Nilsson, R. H., Ryberg, M., Kristiansson, E., Abarenkov, K., Larsson, K. H., & Kõljalg, U. (2006). Taxonomic reliability of DNA sequences in public sequence databases: A fungal perspective. PLoS ONE, 1, e59. https://doi.org/10.1371/journal.pone.0000059
Oliveros, J. C. (2015) Venny. An interactive tool for comparing lists with Venn's diagrams. Retrieved from http://bioinfogp.cnb.csic.es/tools/venny/index.html
Opel, K. L., Chung, D., & McCord, B. R. (2010). A study of PCR inhabitation mechanisms using real time PCR. Journal of Forensic Science, 55, 25-33. https://doi.org/10.1111/j.1556-4029.2009.01245.x
Patti, F. P., & Gambi, M. C. (2001). Phylogeography of the invasive polychaete Sabella spallanzanii (Sabellidae) based on the nucleotide sequence of internal transcribed spacer 2 (ITS2) of nuclear rDNA. Marine Ecology Progress Series, 215, 169-177. https://doi.org/10.1017/S0025315417000261P
Plough, L. V., Ogburn, M. B., Fitzgerald, C. L., Geranio, R., Marafino, G. A., & Richie, K. D. (2018). Environmental DNA analysis of river herring in Chesapeake Bay: A powerful tool for monitoring threatened keystone species. PLoS ONE, 13, e0205578. https://doi.org/10.1371/journal.pone.0205578
Pochon, X., Bott, N., Smith, K. F., & Wood, S. A. (2013). Evaluating detection limits of Next Generation Sequencing for the surveillance and monitoring of international marine pests. PLoS ONE, 8, e73935. https://doi.org/10.1371/journal.pone.0073935
Pochon, X., Zaiko, A., Fletcher, L. M., Laroche, O., & Wood, S. A. (2017). Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications. PLoS ONE, 12, e0187636. https://doi.org/10.1371/journal.pone.0187636
Pregler, K. C., Vokoun, J. C., Jensen, T., & Hagstrom, N. (2015). Using multimethod occupancy estimation models to quantify gear differences in detection probabilities: Is backpack electrofishing missing occurences for a species of concern? Transactions of the American Fishing Society, 141, 89-95. https://doi.org/10.1080/00028487.2014.968291
R Development Core Team (2014). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. http://www.R-project.org
Ratnasingham, S., & Hebert, P. D. N. (2007). BOLD: The barcode of life data system (www.barcodinglife.org). Molecular Ecology Notes, 7, 355-364. https://doi.org/10.1111/j.1471-8286.2006.01678.x
Read, G. B., Inglis, G. J., Stratford, P., & Ahyong, S. T. (2011). Arrival of the alien fanworm Sabella spallanzanii (Gmelin, 1791) (Polychaeta: Sabellidae) in two New Zealand harbours. Aquatic Invasion, 6, 273-279. https://doi.org/10.3391/ai.2011.6.3.04
Robin, X., Turk, N., Hainard, A., Tiberti, N., Lisacek, F., Sanchez, J.-C., & Müller, M. (2011). pROC: An open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics, 12, 77. https://doi.org/10.1186/1471-2105-12-77
Rognes, T., Flouri, T., Nichols, B., Quince, C., & Mahé, F. (2016). VSEARCH: A versatile open source tool for metagenomics. PeerJ, 4, e2584. https://doi.org/10.7717/peerj.2584
Sigsgaard, E. E., Carl, H., Møller, P. R., & Thomsen, P. F. (2015). Monitoring the near-extinct European weather loach in Denmark based on environmental DNA from water samples. Biological Conservation, 183, 46-52. https://doi.org/10.1016/j.biocon.2014.11.023
Smith, K. F., Wood, S. A., Mountfort, D., & Cary, S. C. (2012). Development of a real-time PCR assay for the detection of the invasive clam, Corbula amurensis, in environmental samples. Journal of Experimental and Marine Biology and Ecology, 412, 52-57. https://doi.org/10.1016/j.jembe.2011.10.021
Taberlet, P., Bonin, A., Zinger, L., & Coissac, E. (Eds) (2018). Environmental DNA for biodiversity research and monitoring. Oxford, UK: Oxford University Press.
Tait, L., Inglis, G., & Seaward, K. (2018). Enhancing passive sampling tools for detecting marine bioinvasions. Marine Pollution Bulletin, 128, 41-50. https://doi.org/10.1016/j.marpolbul.2018.01.015
Takahara, T., Minamoto, T., & Doi, H. (2013). Using environmental DNA to estimate the distribution of an invasive fish species in ponds. PLoS ONE, 8, e56584. https://doi.org/10.1371/journal.pone.0056584
Te, S. H., Chen, E. Y., & Gin, K. Y. (2015). Comparison of quantitative PCR and droplet digital PCR multiplex assays for two genera of bloom-forming cyanobacteria, Cylindrospermopsis and Microcystis. Applied and Environmental Microbiology, 81(5203-5211), 5203-5211. https://doi.org/10.1128/AEM.00931-15
Thomsen, P. F., Kielgast, J., Iversen, L. L., Møller, P. R., Rasmussen, M., & Willerslev, E. (2012). Detection of a diverse marine fish fauna using environmental DNA from seawater samples. PLoS ONE, 7, e41732. https://doi.org/10.1371/journal.pone.0041732
von Ammon, U., Wood, S. A., Laroche, O., Zaiko, A., Tait, L., Lavery, S., … Pochon, X. (2017). The impact of artificial surfaces on marine bacterial and eukaryotic biofouling assemblages: A high-throughput sequencing analysis. Marine Environmental Research, 133, 57-66. https://doi.org/10.1016/j.marenvres.2017.12.003
Wang, Q., Garrity, G. M., Tiedje, J. M., & Cole, J. R. (2007). Naive bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology, 73, 5261-5267. https://doi.org/10.1128/AEM.00062-07
Wangensteen, O. S., Palacín, C., Guardiola, M., & Turon, X. (2018). DNA metabarcoding of littoral hard-bottom communities: High diversity and database gaps revealed by two molecular markers. PeerJ, 6, e4705. https://doi.org/10.7717/peerj.4705
Wilcox, T. M., McKelvey, K. S., Young, M. K., Jane, S. F., Lowe, W. H., Whiteley, A. R., & Schwartz, M. K. (2013). Robust detection of rare species using environmental DNA: The importance of primer specificity. PLoS ONE, 8(3), e59520. https://doi.org/10.1371/journal.pone.0059520
Wood, S. A., Pochon, X., Ming, W., von Ammn, U., Woods, C., Carter, M., … Zaiko, A. (2018). Considerations for incorporating real-time PCR assays into routine marine biosecurity surveillance programmes: A case study targeting the Mediterranean fanworm (Sabella spallanzanii) and club tunicate (Styela clava). Genome, 62, 137-146. https://doi.org/10.1139/gen-2018-0021
Wood, S. A., Smith, K. F., Banks, J. C., Tremblay, L., Rhodes, L., Mountfort, D., … & Pochon, X. (2013). Molecular genetic tools for environmental monitoring of New Zealand’s aquatic habitats, past, present and the future. New Zealand Journal of Marine and Freshwater Research, 47, 90-119.
Wood, S. A., Zaiko, A., Richter, I., Inglis, G., & Pochon, X. (2017). Development of a real-time Polymerase Chain Reaction assay for the detection of the invasive Mediterranean fanworm, Sabella spallanzanii, in environmental samples. Environmental Science and Pollution Research, 24, 17373-17382. https://doi.org/10.1007/s11356-017-9357-y
Zaiko, A., Schimanski, K., Pochon, X., Hopkins, G. A., Goldstien, S., Floerl, O., & Wood, S. A. (2016). Metabarcoding improves detection of eukaryotes from early biofouling communities: Implications for pest monitoring and pathway management. Biofouling, 32, 671-684. https://doi.org/10.1080/08927014.2016.1186165
Zhan, A., Hulák, M., Sylvester, F., Huang, X., Adebayo, A. A., Abbott, C. L., … MacIsaac, H. J. (2013). High sensitivity of 454 pyrosequencing for detection of rare species in aquatic communities. Methods in Ecology and Evolution, 4, 558-565. https://doi.org/10.1111/2041-210X.12037
Zinger, L., Bonin, A., Alsos, I. G., Bálint, M., Bik, H., Boyer, F., … Taberlet, P. (2019). DNA metabarcoding-Need for robust experimental designs to draw sound ecological conclusions. Molecular Ecology, 28(8), 1857-1862. https://doi.org/10.1111/mec.15060