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Figures & Tables

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.
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.

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 In contrast, the outdated in-house curated SILVA v111 and the current v138 consisted of variable strings and/or outdated classification (indicated in red) and missing relevant to nematodes taxonomic information (blank cells).
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 In contrast, the outdated in-house curated SILVA v111 and the current v138 consisted of variable strings and/or outdated classification (indicated in red) and missing relevant to nematodes taxonomic information (blank cells).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.
Number of available 18S reference sequences for Nematoda at the order level within SILVA v111, v138, and 18S-NemaBase.

List of primers depicted in Figure 1_ Details include the primer name, author, and citation_

PrimerAuthorCitation
F04 - R22Fonseca et al., 2019Fonseca, 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_R22Blaxter et al., 1998Blaxter, 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- 1132rmodGeisen et al., 2019Geisen, 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-NSR951Mangot et al., 2013Mangot, 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 - MMSRSidker et al., 2020Sikder, 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 - EcoRWaeyenberge et al., 2020Waeyenberge, 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 - 18SILVOmidRWaeyenberge et al., 2020Waeyenberge, 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- 2646RHolterman et al., 2006Holterman 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 - 18Sr2bPorazinska et al., 2009 and Sapkota et al., 2015Porazinska, 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 - 18Sr2boptWaeyenberge et al., 2020Waeyenberge, 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-1631Müller et al., 2019 and Starke et al., 2016Müller, 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 - 18Sr2bPorazinska et al., 2009Porazinska, 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- EukBrAmaral-Zettler et al., 2009 and Caporaso et al., 2012Amaral-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

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_

Number of Taxa
OrderGenusV111V13818S-NemaBase
RhabditidaMeloidogyne109218238
Caenorhabditis59228213
Bursaphelenchus77116117
TrichinellidaTrichinella12564563
Capillaria11616
Aonchotheca03030
DorylaimidaXiphinema148137137
Longidorus479292
Enchodelus91212
EnoplidaHalalaimus434343
Oxystomina262626
Oncholaimus222525
TriplonchidaParatrichodorus345757
Trichodorus264646
Tripyla213030
DesmodoridaLeptonemella11919
Robbea1099
Laxus499
PlectidaPlectus152424
Chronogaster577
Camacolaimus255
MonhysteridaEumonhystera31111
Monhystera388
Daptonema81010
MononchidaMylonchulus272727
Mononchus71111
Clarkus666
AraeolaimidaSabatieria899
Axonolaimus444
Ascolaimus444
MermithidaIsomermis999
Mermis244
Pheromermis022
DesmodoridaDesmoscolex122
Cyartonema111

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_

FamilyGenusSpeciesv111v138-unmodifiedv138-modified18S-NemaBase18S-NemaBase-supplemented
TobrilidaeBrevitobrilusBrevitobrilus sp. Female SALCI Border00003
TobrilidaeEpitobrilusEpitobrilus sp. Male SALCI Border00004
TobrilidaeEpitobrilusEpitobrilus stefanskii00550
TobrilidaeNeotobrilusNeotobrilus sp. Female SALCI Island00001
TobrilidaeSemitobrilusSemitobrilus cf. pellucidus 1 JH-201400330
TobrilidaeTobrilusTobrilus cf. gracilis 2 JH-201400110
TobrilidaeTobrilusTobrilus gracilis60000
TobrilidaeTobrilusTobrilus pellucidus00111
TobrilidaeTobrilusTobrilus sp. Female SALCI Island00001
TobrilidaeTobrilusTobrilus sp. ZQZ-2010a70033
Total ASV/species13/2010/413/513/6
Nematode_environmental sample001400
Nematode_uncultured eukaryote10300
Uncultured nematode290000
BCP Clade0178000
DOI: https://doi.org/10.2478/jofnem-2023-0006 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X
Language: English
Submitted on: Aug 1, 2022
Published on: Apr 21, 2023
Published by: Society of Nematologists, Inc.
In partnership with: Paradigm Publishing Services
Publication frequency: 1 issue per year

© 2023 Kaitlin Gattoni, Eli M. S. Gendron, Rebeca Sandoval-Ruiz, Abigail Borgemeier, J. Parr McQueen, Rachel M. Shepherd, Dieter Slos, Thomas O. Powers, Dorota L. Porazinska, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution 4.0 License.