Figure 1
Genetic location of primers commonly used in nematode metabarcoding. Depicted is the entire rRNA gene with close ups of 18S rRNA gene with possible primers aligned below it. The 18S rRNA primers were aligned with Caenorhabditis elegans SSU (GenBank accession number: AY268117, X03680 and MN519140) to define the base pair locations (indicated in grey). References for primer sets available in Supplemental Table 1.
Table 1
The identities and number of 3 most populated genera across nematode orders in SILVA v111, SILVA V138, and 18S-NemaBase. The list is sorted from the largest to smallest number of total representative sequences.
| Order | Genus | V111 | V138 | 18S-NemaBase |
|---|---|---|---|---|
| Rhabditida | Meloidogyne | 109 | 218 | 238 |
| Caenorhabditis | 59 | 228 | 213 | |
| Bursaphelenchus | 77 | 116 | 117 | |
| Trichinellida | Trichinella | 12 | 564 | 563 |
| Capillaria | 1 | 16 | 16 | |
| Aonchotheca | 0 | 30 | 30 | |
| Dorylaimida | Xiphinema | 148 | 137 | 137 |
| Longidorus | 47 | 92 | 92 | |
| Enchodelus | 9 | 12 | 12 | |
| Enoplida | Halalaimus | 43 | 43 | 43 |
| Oxystomina | 26 | 26 | 26 | |
| Oncholaimus | 22 | 25 | 25 | |
| Triplonchida | Paratrichodorus | 34 | 57 | 57 |
| Trichodorus | 26 | 46 | 46 | |
| Tripyla | 21 | 30 | 30 | |
| Desmodorida | Leptonemella | 1 | 19 | 19 |
| Robbea | 10 | 9 | 9 | |
| Laxus | 4 | 9 | 9 | |
| Plectida | Plectus | 15 | 24 | 24 |
| Chronogaster | 5 | 7 | 7 | |
| Camacolaimus | 2 | 5 | 5 | |
| Monhysterida | Eumonhystera | 3 | 11 | 11 |
| Monhystera | 3 | 8 | 8 | |
| Daptonema | 8 | 10 | 10 | |
| Mononchida | Mylonchulus | 27 | 27 | 27 |
| Mononchus | 7 | 11 | 11 | |
| Clarkus | 6 | 6 | 6 | |
| Araeolaimida | Sabatieria | 8 | 9 | 9 |
| Axonolaimus | 4 | 4 | 4 | |
| Ascolaimus | 4 | 4 | 4 | |
| Mermithida | Isomermis | 9 | 9 | 9 |
| Mermis | 2 | 4 | 4 | |
| Pheromermis | 0 | 2 | 2 | |
| Desmodorida | Desmoscolex | 1 | 2 | 2 |
| Cyartonema | 1 | 1 | 1 |
Figure 2
An example of variation of taxonomic categories and ranks resulting in variable strings for Meloidogyne arenaria across the databases. The curated 18S-NemaBase includes standardized taxonomic ranks consisting of 13 Nematoda relevant categories (top row) and currently accepted classification as adopted by the WoRMS database. In contrast, the outdated in-house curated SILVA v111 and the current v138 consisted of variable strings and/or outdated classification (indicated in red) and missing relevant to nematodes taxonomic information (blank cells).
Figure 3
Number of available 18S reference sequences for Nematoda at the order level within SILVA v111, v138, and 18S-NemaBase.
Table 2
Tobrilidae species from the Western Nebraska Sandhills dataset assigned by v111, v138, and 18S-NemaBase, and 18S-NemaBase-supplemented databases. The numbers represent how many distinct ASVs assigned to that species.
| Family | Genus | Species | v111 | v138-unmodified | v138-modified | 18S-NemaBase | 18S-NemaBase-supplemented |
|---|---|---|---|---|---|---|---|
| Tobrilidae | Brevitobrilus | Brevitobrilus sp. Female SALCI Border | 0 | 0 | 0 | 0 | 3 |
| Tobrilidae | Epitobrilus | Epitobrilus sp. Male SALCI Border | 0 | 0 | 0 | 0 | 4 |
| Tobrilidae | Epitobrilus | Epitobrilus stefanskii | 0 | 0 | 5 | 5 | 0 |
| Tobrilidae | Neotobrilus | Neotobrilus sp. Female SALCI Island | 0 | 0 | 0 | 0 | 1 |
| Tobrilidae | Semitobrilus | Semitobrilus cf. pellucidus 1 JH-2014 | 0 | 0 | 3 | 3 | 0 |
| Tobrilidae | Tobrilus | Tobrilus cf. gracilis 2 JH-2014 | 0 | 0 | 1 | 1 | 0 |
| Tobrilidae | Tobrilus | Tobrilus gracilis | 6 | 0 | 0 | 0 | 0 |
| Tobrilidae | Tobrilus | Tobrilus pellucidus | 0 | 0 | 1 | 1 | 1 |
| Tobrilidae | Tobrilus | Tobrilus sp. Female SALCI Island | 0 | 0 | 0 | 0 | 1 |
| Tobrilidae | Tobrilus | Tobrilus sp. ZQZ-2010a | 7 | 0 | 0 | 3 | 3 |
| Total ASV/species | 13/2 | 0 | 10/4 | 13/5 | 13/6 | ||
| Nematode_environmental sample | 0 | 0 | 14 | 0 | 0 | ||
| Nematode_uncultured eukaryote | 1 | 0 | 3 | 0 | 0 | ||
| Uncultured nematode | 29 | 0 | 0 | 0 | 0 | ||
| BCP Clade | 0 | 178 | 0 | 0 | 0 |
Supplemental Table 1
List of primers depicted in Figure 1. Details include the primer name, author, and citation.
| Primer | Author | Citation |
|---|---|---|
| F04 - R22 | Fonseca et al., 2019 | Fonseca, V. G., Carvalho, G. R., Sung, W., Johnson, H. F., Power, D. M., Neill, S. P., Packer, M., Blaxter, M. L., Lambshead, P. J. D., Thomas, W. K., and Creer, S. 2010. Second-generation environmental sequencing unmasks marine metazoan biodiversity. Nature Communications 1(98). http://https://dol/10.1038/ncomms1095 |
| SSU_F04 - SSU_R22 | Blaxter et al., 1998 | Blaxter, M. L., De Ley, P., Garey, J. R., Liu, L. X., Scheldeman, P., Vierstraete, A., Vanfleteren, J. R., Mackey, L. Y., Dorris, M., Frisse, L. M., Vida, J. T., and Thomas, W. K. 1998. A molecular evolutionary framework for the phylum Nematoda. Nature 392:71-75. http://https://dol/10.1038/32160 |
| 3NDf- 1132rmod | Geisen et al., 2019 | Geisen, S., Snoek, L. B., ten Hooven, F. C., Duyts, H., Kostenko, O., Bloem, J., Martens, H., Quist, C. W., Helder, J. A., and van den Putten, W. H. 2018. Integrating quantitative morphological and qualitative molecular methods to analyse soil nematode community responses to plant range expansion. Methods In Ecology and Evolution 9:1366-1378. http://https://dol/10.1111/2041 -210x.12999 |
| Ek-NSF573 - EK-NSR951 | Mangot et al., 2013 | Mangot, J.-F., Domaizon, I., Taib, N., Marouni, N., Duffaud, E., Bronner, G., and Debroas, D. 2013. Short-term dynamics of diversity patterns: evidence of continual reassembly within lacustrine small eukaryotes. Environmental Microbiology 15: 1745-1758. http://https://dol.org/10.1111/1462-2920.12065 |
| MMSF - MMSR | Sidker et al., 2020 | Sikder, M. M., Vestergård, M., Sapkota, R., Kyndt, T., and Nicolaisen, M. 2020. Evaluation of Metabarcoding Primers for Analysis of Soil Nematode Communities. Diversity 12(388). http://https://dol.org/10.3390/d12100388 |
| EcoF - EcoR | Waeyenberge et al., 2020 | Waeyenberge, L., Sutter, N. D., Viaene, N., and Haegeman, A. 2019. New Insights Into nematode DNA-metabarcoding as revealed by the characterization of artificial and spiked nematode communities. Diversity 11 (4):52. http://https://dol.org/10.3390/d11040052 |
| 18SILVOmidF - 18SILVOmidR | Waeyenberge et al., 2020 | Waeyenberge, L., Sutter, N. D., Viaene, N., and Haegeman, A. 2019. New Insights Into nematode DNA-metabarcoding as revealed by the characterization of artificial and spiked nematode communities. Diversity 11 (4):52. http://https://dol.org/10.3390/d11040052 |
| 1813F- 2646R | Holterman et al., 2006 | Holterman M., van der Wurff A., van den Elsen S., van Megen H., Bongers T., Holovachov O., Bakker J., and Helder J., 2006. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biology and Evolution 23:1792-1800. http://https://doi/10.1093/molbev/msl044 |
| NemF - 18Sr2b | Porazinska et al., 2009 and Sapkota et al., 2015 | Porazinska, D. L, Giblin-Davis, R. M., Faller, L, Farmerie, W., Kanzaki, N., Morris, K., Powers, T. O., Tucker, A. E., Sung, W., and Thomas, W. K. 2009. Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversity. Molecular Ecology Resoures 9:1439-50. http://https://doi.Org/10.1111/j.1755-0998.2009.02611; Sapkota, R., and Nicolaisen, M. 2015. High-throughput sequencing of nematode communities from total soil DNA extractions. BMC Ecology 12(15) http://https://doi.org/10.1186/s12898-014-0034-4 |
| NemFopt - 18Sr2bopt | Waeyenberge et al., 2020 | Waeyenberge, L., Sutter, N. D., Viaene, N., and Haegeman, A. 2019. New insights into nematode DNA-metabarcoding as revealed by the characterization of artificial and spiked nematode communities. Diversity 11 (4):52. http://https://dol.org/10.3390/d11040052 |
| F-1183 - R-1631 | Müller et al., 2019 and Starke et al., 2016 | Müller, C. A., Pereira, L. D., Lopes, C., Cares, J., Borges, L. G. D., Giongo, A., Graeff-Teixeira, C., and Morassutti. 2019. Meiofaunal diversity In the Atlantic Forest soil: A quest for nematodes in a native reserve using eukaryotic metabarcoding analysis. Forest Ecology and Management 453. http://https://doi.Org/10.1016/j.foreco.2019.117591; Starke, R., Kermer, R., Ullmann-Zeunert, L., Baldwin, I. T., Seifert, J., Bastida, F., von Bergen, M., and Jehmlich, N. 2016. Bacteria dominate the short-term assimilation of plant-derived N in soil. Soil Biology and Biochemistry 96:30-38. http://https://doi.Org/10.1016/j.soilbio.2016.01.009 |
| NF1 - 18Sr2b | Porazinska et al., 2009 | Porazinska, D. L, Giblin-Davis, R. M., Faller, L, Farmerie, W., Kanzaki, N., Morris, K., Powers, T. O., Tucker, A. E., Sung, W., and Thomas, W. K. 2009. Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversity. Molecular Ecology Resources 9:1439-50. http://https://doi.Org/10.1111/j.1755-0998.2009.02611 |
| 1391f- EukBr | Amaral-Zettler et al., 2009 and Caporaso et al., 2012 | Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W., and Huse, S. M. 2009. A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PLoS One 4(7): 1 —9. http://https://doi.org/10.1371/journal.pone.0006372; Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S. M., Betley, J., Fraser, L, Bauer, M., Gormley, N., Gilbert, J. A., Smith, G., and Knight, R. 2012. Ultra-high-throughput microbial community analysis on the lllumina HiSeq and MiSeq platforms. ISME Journal 6(8):1621-1624. http://https://doi.Org/10.1038/ismej.2012.8 |