Over 70% of nematode species are free-living. They are found in a wide variety of habitats and are essential for maintaining microenvironments. However, studies on nematodes have mainly focused on parasites and terrestrial free-living. Free-living nematodes in aquatic ecosystems represent 50% of all nematode species but are poorly understood. Recently, studies of marine free-living nematodes have focused on their potential to address nematode phylogeny questions and explore the evolution of nematode parasitism and adaptation to extreme environments. My Ph.D. aims to sequence the genome of a marine free-living nematode to reconstruct nematode phylogeny and compare it with published nematode genomes to investigate nematode genome diversity. During the sampling process, I analyzed the nematode community around the northern coast of Taiwan. It is important to note that marine nematode species are not amenable to axenic culture under laboratory conditions. To alleviate this issue, I utilized multiple displacement amplification (MDA) and Smartseq2 to amplify nanograms of genomic DNA and mRNA from one individual, respectively. We evaluated the bias of this protocol using Caenorhabditis elegans. While reduced genome coverage was detected in repetitive regions, we generated assemblies that covered 98% of the reference genome using long-read sequences produced with Oxford Nanopore Technologies (ONT). Annotation of the sequenced transcriptome and available assemblies showed that gene predictions were more precise, comprehensive, and contained fewer false positives than de novo transcriptome assembly methods. Our results demonstrate the robustness of MDA in combination with ONT, paving the way for the study of genome diversity in the phylum Nematoda and beyond.

Ahmed, M., Roberts, N. G., Adediran, F., Smythe, A. B., Kocot, K. M., & Holovachov, O. (2022). Phylogenomic Analysis of the Phylum Nematoda: Conflicts and Congruences With Morphology, 18S rRNA, and Mitogenomes. Frontiers in Ecology and Evolution, 9. doi:10.3389/fevo.2021.769565
Ahmed, M. S., M.; Prior, T., Karssen, G.; & Back, M. (2015). Nematode taxonomy: From morphology to metabarcoding. SOIL Discussions, 2(2), 1175–1220. doi:https://doi.org/10.5194/soild-2-1175-2015
Andrew D. Young, J. P. G. (2020). Phylogenomics—principles, opportunities and pitfalls of big‐data phylogenetics. Systematic Entomology, 45, 225–247 doi:10.1111/syen.12406
Andrews, S. (2010). FastQC: A Quality Control Tool for High Throughput Sequence Data [Online].
Antil, S., Abraham, J. S., Sripoorna, S., Maurya, S., Dagar, J., Makhija, S., . . . Toteja, R. (2023). DNA barcoding, an effective tool for species identification: a review. Mol Biol Rep, 50(1), 761-775. doi:10.1007/s11033-022-08015-7
Arora, D., Hernandez, A. G., Walden, K. K. O., Fields, C. J., & Yan, G. (2023). First Draft Genome Assembly of Root-Lesion Nematode Pratylenchus scribneri Generated Using Long-Read Sequencing. Int J Mol Sci, 24(8). doi:10.3390/ijms24087311
Arroyo Muhr, L. S., Lagheden, C., Hassan, S. S., Kleppe, S. N., Hultin, E., & Dillner, J. (2020). De novo sequence assembly requires bioinformatic checking of chimeric sequences. PLoS One, 15(8), e0237455. doi:10.1371/journal.pone.0237455
Bai, X., Adams, B. J., Ciche, T. A., Clifton, S., Gaugler, R., Kim, K. S., . . . Grewal, P. S. (2013). A lover and a fighter: the genome sequence of an entomopathogenic nematode Heterorhabditis bacteriophora. PLoS One, 8(7), e69618. doi:10.1371/journal.pone.0069618
Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., . . . Pevzner, P. A. (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol, 19(5), 455-477. doi:10.1089/cmb.2012.0021
Bardgett, R. D., & van der Putten, W. H. (2014). Belowground biodiversity and ecosystem functioning. Nature, 515(7528), 505-511. doi:10.1038/nature13855
Bernt, M., Donath, A., Juhling, F., Externbrink, F., Florentz, C., Fritzsch, G., . . . Stadler, P. F. (2013). MITOS: improved de novo metazoan mitochondrial genome annotation. Mol Phylogenet Evol, 69(2), 313-319. doi:10.1016/j.ympev.2012.08.023
Bik, H. M., Lambshead, P. J., Thomas, W. K., & Lunt, D. H. (2010). Moving towards a complete molecular framework of the Nematoda: a focus on the Enoplida and early-branching clades. BMC Evol Biol, 10, 353. doi:10.1186/1471-2148-10-353
Blainey, P. C., & Quake, S. R. (2011). Digital MDA for enumeration of total nucleic acid contamination. Nucleic Acids Res, 39(4), e19. doi:10.1093/nar/gkq1074
Blaxter, D. L. (2002). Systematic position and phylogeny (I. L. D. (ed) Ed.): Harwood Acad Publ.
Blaxter, M. (2011). Nematodes: the worm and its relatives. PLoS Biol, 9(4), e1001050. doi:10.1371/journal.pbio.1001050
Blaxter, M. L., De Ley, P., Garey, J. R., Liu, L. X., Scheldeman, P., Vierstraete, A., . . . Thomas, W. K. (1998). A molecular evolutionary framework for the phylum Nematoda. Nature, 392(6671), 71-75. doi:10.1038/32160
Bogale, M., Baniya, A., & DiGennaro, P. (2020). Nematode Identification Techniques and Recent Advances. Plants (Basel), 9(10). doi:10.3390/plants9101260
Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114-2120. doi:10.1093/bioinformatics/btu170
Boore, J. L. (1999). Animal mitochondrial genomes. Nucleic Acids Res, 27(8), 1767-1780. doi:10.1093/nar/27.8.1767
Bowman, J. P. (2006). The Marine Clade of the Family Flavobacteriaceae: The Genera Aequorivita, Arenibacter, Cellulophaga, Croceibacter, Formosa, Gelidibacter, Gillisia, Maribacter, Mesonia, Muricauda, Polaribacter, Psychroflexus, Psychroserpens, Robiginitalea, Salegentibacter, Tenacibaculum, Ulvibacter, Vitellibacter and Zobellia. In M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer, & E. Stackebrandt (Eds.), The Prokaryotes: Volume 7: Proteobacteria: Delta, Epsilon Subclass (pp. 677-694). New York, NY: Springer New York.
Bruna, T., Hoff, K. J., Lomsadze, A., Stanke, M., & Borodovsky, M. (2021). BRAKER2: automatic eukaryotic genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database. NAR Genom Bioinform, 3(1), lqaa108. doi:10.1093/nargab/lqaa108
Buchfink, B., Reuter, K., & Drost, H.-G. (2021). Sensitive protein alignments at tree-of-life scale using DIAMOND. Nature Methods, 18(4), 366-368. doi:10.1038/s41592-021-01101-x
The C. elegans Sequencing Consortium. (1998). Genome sequence of the nematode C. elegans: a platform for investigating biology. Science, 282(5396), 2012-2018. doi:10.1126/science.282.5396.2012
Cai, L., Fu, S., Zhou, X., Tseng, L.-C., & Hwang, J.-S. (2020). Benthic meiofauna with emphasis on nematode assemblage response to environmental variation in the intertidal zone of the Danshuei River estuary, northwest Taiwan. Ecological Research, 35(5), 857-870. doi:https://doi.org/10.1111/1440-1703.12159
Calus, S. T., Ijaz, U. Z., & Pinto, A. J. (2018). NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform. Gigascience, 7(12). doi:10.1093/gigascience/giy140
Carlton, P. M., Davis, R. E., & Ahmed, S. (2022). Nematode chromosomes. Genetics, 221(1). doi:10.1093/genetics/iyac014
Christoph Dieterich, R. J. S. (2009). How to become a parasite - lessons from the genomes of nematodes. Trends in Genetics, 25(5), 203-209. doi:https://doi.org/10.1016/j.tig.2009.03.006
Coate, J. E., & Doyle, J. J. (2015). Variation in transcriptome size: are we getting the message? Chromosoma, 124(1), 27-43. doi:10.1007/s00412-014-0496-3
Coghlan, A., Tsai, I., & Berriman, M. (2018). Creation of a comprehensive repeat library for a newly sequenced parasitic worm genome. avaliable at Protocol Exchange doi:https://doi.org/10.1038/protex.2018.054
Coghlan, A., Tyagi, R., Cotton, J. A., Holroyd, N., Rosa, B. A., Tsai, I. J., . . . International Helminth Genomes, C. (2019). Comparative genomics of the major parasitic worms. Nature Genetics, 51(1), 163-174. doi:10.1038/s41588-018-0262-1
D'Hondt, A. S., Stock, W., Blommaert, L., Moens, T., & Sabbe, K. (2018). Nematodes stimulate biomass accumulation in a multispecies diatom biofilm. Mar Environ Res, 140, 78-89. doi:10.1016/j.marenvres.2018.06.005
De Ley, P. (2006). A quick tour of nematode diversity and the backbone of nematode phylogeny. WormBook, 1-8. doi:10.1895/wormbook.1.41.1
De Vivo, M., Lee, H.-H., Huang, Y.-S., Dreyer, N., Fong, C.-L., de Mattos, F. M. G., . . . Tsai, I. J. (2022). Utilisation of Oxford Nanopore sequencing to generate six complete gastropod mitochondrial genomes as part of a biodiversity curriculum. Scientific Reports, 12(1), 9973. doi:10.1038/s41598-022-14121-0
Dean, F. B., Nelson, J. R., Giesler, T. L., & Lasken, R. S. (2001). Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res, 11(6), 1095-1099. doi:10.1101/gr.180501
Deiner, K., Bik, H. M., Machler, E., Seymour, M., Lacoursiere-Roussel, A., Altermatt, F., . . . Bernatchez, L. (2017). Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Mol Ecol, 26(21), 5872-5895. doi:10.1111/mec.14350
Deleye, L., Tilleman, L., Vander Plaetsen, A. S., Cornelis, S., Deforce, D., & Van Nieuwerburgh, F. (2017). Performance of four modern whole genome amplification methods for copy number variant detection in single cells. Sci Rep, 7(1), 3422. doi:10.1038/s41598-017-03711-y
Denton, J. F., Lugo-Martinez, J., Tucker, A. E., Schrider, D. R., Warren, W. C., & Hahn, M. W. (2014). Extensive error in the number of genes inferred from draft genome assemblies. PLoS Comput Biol, 10(12), e1003998. doi:10.1371/journal.pcbi.1003998
di Montanara, A. C., Baldrighi, E., Franzo, A., Catani, L., Grassi, E., Sandulli, R., & Semprucci, F. (2022). Free-living nematodes research: State of the art, prospects, and future directions. A bibliometric analysis approach. Ecological Informatics, 72, 101891. doi:https://doi.org/10.1016/j.ecoinf.2022.101891
Dillman, A. R., Mortazavi, A., & Sternberg, P. W. (2012). Incorporating genomics into the toolkit of nematology. J Nematol, 44(2), 191-205. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/23482088
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578471/pdf/191.pdf
Dobin, A., & Gingeras, T. R. (2015). Mapping RNA-seq Reads with STAR. Curr Protoc Bioinformatics, 51, 11 14 11-11 14 19. doi:10.1002/0471250953.bi1114s51
Doyle, S. R., Sankaranarayanan, G., Allan, F., Berger, D., Jimenez Castro, P. D., Collins, J. B., . . . Holroyd, N. (2019). Evaluation of DNA Extraction Methods on Individual Helminth Egg and Larval Stages for Whole-Genome Sequencing. Front Genet, 10, 826. doi:10.3389/fgene.2019.00826
Eccles, D., Chandler, J., Camberis, M., Henrissat, B., Koren, S., Le Gros, G., & Ewbank, J. J. (2018). De novo assembly of the complex genome of Nippostrongylus brasiliensis using MinION long reads. BMC Biol, 16(1), 6. doi:10.1186/s12915-017-0473-4
Edgar, R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics, 26(19), 2460-2461. doi:10.1093/bioinformatics/btq461
Ekblom, R., & Galindo, J. (2011). Applications of next generation sequencing in molecular ecology of non-model organisms. Heredity (Edinb), 107(1), 1-15. doi:10.1038/hdy.2010.152
Emms, D. M., & Kelly, S. (2015). OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol, 16(1), 157. doi:10.1186/s13059-015-0721-2
Faulk, C. (2023). De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing. Nucleic Acids Res, 51(1), 17-28. doi:10.1093/nar/gkac510
Feng, B., Zhou, L., & Tang, J. (2017). Ancestral Genome Reconstruction on Whole Genome Level. Curr Genomics, 18(4), 306-315. doi:10.2174/1389202918666170307120943
Ferguson, D. K., Li, C., Chakraborty, A., Gittins, D. A., Fowler, M., Webb, J., . . . Hubert, C. R. J. (2023). Multi-year seabed environmental baseline in deep-sea offshore oil prospective areas established using microbial biodiversity. Marine Pollution Bulletin, 194, 115308. doi:https://doi.org/10.1016/j.marpolbul.2023.115308
Flynn, J. M., Hubley, R., Goubert, C., Rosen, J., Clark, A. G., Feschotte, C., & Smit, A. F. (2020). RepeatModeler2 for automated genomic discovery of transposable element families. Proc Natl Acad Sci U S A, 117(17), 9451-9457. doi:10.1073/pnas.1921046117
Gingold, R., Moens, T., & Rocha-Olivares, A. (2013). Assessing the Response of Nematode Communities to Climate Change-Driven Warming: A Microcosm Experiment. PLoS One, 8(6), e66653. doi:10.1371/journal.pone.0066653
Goodwin, S., McPherson, J. D., & McCombie, W. R. (2016). Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet, 17(6), 333-351. doi:10.1038/nrg.2016.49
Haas, B. (2020). TransDecoder (Find Coding Regions Within Transcripts),. Retrieved from https://github.com/TransDecoder/TransDecoder/wiki
Hård, J., Mold, J. E., Eisfeldt, J., Tellgren-Roth, C., Häggqvist, S., Bunikis, I., . . . Ameur, A. (2023). Long-read whole genome analysis of human single cells. bioRxiv, 2021.2004.2013.439527. doi:10.1101/2021.04.13.439527
Hardison, R. C. (2003). Comparative genomics. PLoS Biol, 1(2), E58. doi:10.1371/journal.pbio.0000058
He, K., Minias, P., & Dunn, P. O. (2021). Long-Read Genome Assemblies Reveal Extraordinary Variation in the Number and Structure of MHC Loci in Birds. Genome Biol Evol, 13(2). doi:10.1093/gbe/evaa270
Hodda, M. (2022). Phylum Nematoda: feeding habits for all valid genera using a new, universal scheme encompassing the entire phylum, with descriptions of morphological characteristics of the stoma, a key, and discussion of the evidence for trophic relationships. Zootaxa, 5114(1), 318-451. doi:10.11646/zootaxa.5114.1.3
Holterman, M., Schratzberger, M., & Helder, J. (2019). Nematodes as evolutionary commuters between marine, freshwater and terrestrial habitats. Biological Journal of the Linnean Society, 128(3), 756-767.
Holterman, M., van der Wurff, A., van den Elsen, S., van Megen, H., Bongers, T., Holovachov, O., . . . Helder, J. (2006). Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown Clades. Mol Biol Evol, 23(9), 1792-1800. doi:10.1093/molbev/msl044
Hongoh, Y., & Toyoda, A. (2011). Whole-genome sequencing of unculturable bacterium using whole-genome amplification. Methods Mol Biol, 733, 25-33. doi:10.1007/978-1-61779-089-8_2
Hotaling, S., Sproul, J. S., Heckenhauer, J., Powell, A., Larracuente, A. M., Pauls, S. U., . . . Frandsen, P. B. (2021). Long Reads Are Revolutionizing 20 Years of Insect Genome Sequencing. Genome Biol Evol, 13(8). doi:10.1093/gbe/evab138
Howe, K. L., Bolt, B. J., Shafie, M., Kersey, P., & Berriman, M. (2017). WormBase ParaSite - a comprehensive resource for helminth genomics. Mol Biochem Parasitol, 215, 2-10. doi:10.1016/j.molbiopara.2016.11.005
Hu, J., Fan, J., Sun, Z., & Liu, S. (2020). NextPolish: a fast and efficient genome polishing tool for long-read assembly. Bioinformatics, 36(7), 2253-2255. doi:10.1093/bioinformatics/btz891
Hu, M., & Gasser, R. B. (2006). Mitochondrial genomes of parasitic nematodes--progress and perspectives. Trends Parasitol, 22(2), 78-84. doi:10.1016/j.pt.2005.12.003
Huang, S., Kang, M., & Xu, A. (2017). HaploMerger2: rebuilding both haploid sub-assemblies from high-heterozygosity diploid genome assembly. Bioinformatics, 33(16), 2577-2579. doi:10.1093/bioinformatics/btx220
Hughes, S., Arneson, N., Done, S., & Squire, J. (2005). The use of whole genome amplification in the study of human disease. Prog Biophys Mol Biol, 88(1), 173-189. doi:10.1016/j.pbiomolbio.2004.01.007
Hyman, B. C., Lewis, S. C., Tang, S., & Wu, Z. (2011). Rampant gene rearrangement and haplotype hypervariation among nematode mitochondrial genomes. Genetica, 139(5), 611-615. doi:10.1007/s10709-010-9531-3
International Helminth Genomes, C. (2019). Comparative genomics of the major parasitic worms. Nat Genet, 51(1), 163-174. doi:10.1038/s41588-018-0262-1
Iqbal, S., & Jones, M. G. K. (2017). Nematodes. In B. Thomas, B. G. Murray, & D. J. Murphy (Eds.), Encyclopedia of Applied Plant Sciences (Second Edition) (pp. 113-119). Oxford: Academic Press.
Juhling, F., Putz, J., Florentz, C., & Stadler, P. F. (2012). Armless mitochondrial tRNAs in Enoplea (Nematoda). RNA Biol, 9(9), 1161-1166. doi:10.4161/rna.21630
Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol, 30(4), 772-780. doi:10.1093/molbev/mst010
Kern, E. M. A., Kim, T., & Park, J. K. (2020). The Mitochondrial Genome in Nematode Phylogenetics. Frontiers in Ecology and Evolution, 8. doi:ARTN 250
10.3389/fevo.2020.00250
Khan, Z., & Kim, Y. H. (2007). A review on the role of predatory soil nematodes in the biological control of plant parasitic nematodes. Applied Soil Ecology, 35(2), 370-379. doi:https://doi.org/10.1016/j.apsoil.2006.07.007
Kiguchi, Y., Nishijima, S., Kumar, N., Hattori, M., & Suda, W. (2021). Long-read metagenomics of multiple displacement amplified DNA of low-biomass human gut phageomes by SACRA pre-processing chimeric reads. DNA Res, 28(6). doi:10.1093/dnares/dsab019
Kikuchi, T., Eves-van den Akker, S., & Jones, J. T. (2017). Genome Evolution of Plant-Parasitic Nematodes. Annu Rev Phytopathol, 55, 333-354. doi:10.1146/annurev-phyto-080516-035434
Kim, J., Kern, E., Kim, T., Sim, M., Kim, J., Kim, Y., . . . Park, J. K. (2017). Phylogenetic analysis of two Plectus mitochondrial genomes (Nematoda: Plectida) supports a sister group relationship between Plectida and Rhabditida within Chromadorea. Mol Phylogenet Evol, 107, 90-102. doi:10.1016/j.ympev.2016.10.010
Kim, S. C., Premasekharan, G., Clark, I. C., Gemeda, H. B., Paris, P. L., & Abate, A. R. (2017). Measurement of copy number variation in single cancer cells using rapid-emulsification digital droplet MDA. Microsyst Nanoeng, 3. doi:10.1038/micronano.2017.18
Kolmogorov, M., Yuan, J., Lin, Y., & Pevzner, P. A. (2019). Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol, 37(5), 540-546. doi:10.1038/s41587-019-0072-8
Ladoukakis, E. D., & Zouros, E. (2017). Evolution and inheritance of animal mitochondrial DNA: rules and exceptions. J Biol Res (Thessalon), 24, 2. doi:10.1186/s40709-017-0060-4
Lai, C. K., Lee, Y. C., Ke, H. M., Lu, M. R., Liu, W. A., Lee, H. H., . . . Tsai, I. J. (2023). The Aphelenchoides genomes reveal substantial horizontal gene transfers in the last common ancestor of free-living and major plant-parasitic nematodes. Mol Ecol Resour. doi:10.1111/1755-0998.13752
Larsen, B. B., Miller, E. C., Rhodes, M. K., & Wiens, J. J. (2017). Inordinate Fondness Multiplied and Redistributed: the Number of Species on Earth and the New Pie of Life. The Quarterly Review of Biology, 92(3), 229-265. doi:10.1086/693564
Lasken, R. S., & Stockwell, T. B. (2007). Mechanism of chimera formation during the Multiple Displacement Amplification reaction. BMC Biotechnol, 7, 19. doi:10.1186/1472-6750-7-19
Lazarova, S., Coyne, D., G. Rodríguez, M., Peteira, B., & Ciancio, A. (2021). Functional Diversity of Soil Nematodes in Relation to the Impact of Agriculture—A Review. Diversity, 13(2), 64. Retrieved from https://www.mdpi.com/1424-2818/13/2/64
Leasi, F., Sevigny, J. L., Laflamme, E. M., Artois, T., Curini-Galletti, M., de Jesus Navarrete, A., . . . Thomas, W. K. (2018). Biodiversity estimates and ecological interpretations of meiofaunal communities are biased by the taxonomic approach. Commun Biol, 1, 112. doi:10.1038/s42003-018-0119-2
Lepere, C., Demura, M., Kawachi, M., Romac, S., Probert, I., & Vaulot, D. (2011). Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations. FEMS Microbiol Ecol, 76(3), 513-523. doi:10.1111/j.1574-6941.2011.01072.x
Lewin, H. A., Richards, S., Lieberman Aiden, E., Allende, M. L., Archibald, J. M., Balint, M., . . . Zhang, G. (2022). The Earth BioGenome Project 2020: Starting the clock. Proc Natl Acad Sci U S A, 119(4). doi:10.1073/pnas.2115635118
Li, H. (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics, 34(18), 3094-3100. doi:10.1093/bioinformatics/bty191
Liao, J. X., Yeh, H. M., & Mok, H. K. (2015). Meiofaunal communities in a tropical seagrass bed and adjacent unvegetated sediments with note on sufficient sample size for determining local diversity indices. Zool Stud, 54, e14. doi:10.1186/s40555-014-0101-8
Liao, W., Hu, J., & Peng, P. a. (2018). Burial of Organic Carbon in the Taiwan Strait. Journal of Geophysical Research: Oceans, 123. doi:10.1029/2018JC014285
Lizhe, C., Huasheng, H., Chaozhong, Z., Shaohua, F., Xiaomei, L., & Yueping, Z. (2001). Species composition and distribution of marine nematode community in the North Taiwan Strait. Acta Oceanologica Sinica(2), 221. Retrieved from http://www.aosocean.com/en/article/id/20010207
Lo, W. S., Roca, M., Dardiry, M., Mackie, M., Eberhardt, G., Witte, H., . . . Lightfoot, J. W. (2022). Evolution and Diversity of TGF-beta Pathways are Linked with Novel Developmental and Behavioral Traits. Mol Biol Evol, 39(12). doi:10.1093/molbev/msac252
Ma, M. Y., Xia, J., Shu, K. X., & Niu, D. K. (2022). Intron losses and gains in the nematodes. Biol Direct, 17(1), 13. doi:10.1186/s13062-022-00328-8
Maggenti, A. R. (1963). Comparative Morphology in Nemic Phylogeny. THE LOWER METAZOA: COMPARATIVE BIOLOGY AND PHYLOGENY, 273-282.
Mahfouz M.M. Abd-Elgawad, T. H. A. (2015). Impact of phytonematodes on agriculture economy.: CAB International.
Majdi, N., & Traunspurger, W. (2015). Free-living nematodes in the freshwater food web: a review. J Nematol, 47(1), 28-44. Retrieved from

Marcais, G., & Kingsford, C. (2011). A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics, 27(6), 764-770. doi:10.1093/bioinformatics/btr011
McLean, F., Berger, D., Laetsch, D. R., Schwartz, H. T., & Blaxter, M. (2018). Improving the annotation of the Heterorhabditis bacteriophora genome. Gigascience, 7(4). doi:10.1093/gigascience/giy034
Meldal, B. H., Debenham, N. J., De Ley, P., De Ley, I. T., Vanfleteren, J. R., Vierstraete, A. R., . . . Lambshead, P. J. (2007). An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa. Mol Phylogenet Evol, 42(3), 622-636. doi:10.1016/j.ympev.2006.08.025
Mikheenko, A., Prjibelski, A., Saveliev, V., Antipov, D., & Gurevich, A. (2018). Versatile genome assembly evaluation with QUAST-LG. Bioinformatics, 34(13), i142-i150. doi:10.1093/bioinformatics/bty266
Moens, T., & Vincx, M. (2000). Temperature, salinity and food thresholds in two brackish-water bacterivorous nematode species: assessing niches from food absorption and respiration experiments. Journal of Experimental Marine Biology and Ecology, 243(1), 137-154. doi:https://doi.org/10.1016/S0022-0981(99)00114-8
Montoliu-Nerin, M., Sanchez-Garcia, M., Bergin, C., Grabherr, M., Ellis, B., Kutschera, V. E., . . . Rosling, A. (2020). Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei. Sci Rep, 10(1), 1303. doi:10.1038/s41598-020-58025-3
Muller, B., Jones, C., & West, S. C. (1990). T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein. Nucleic Acids Res, 18(19), 5633-5636. doi:10.1093/nar/18.19.5633
Ng, W. L., Chen, C. A., Mustafa, S., Soo, C. L., Liao, Y. C., & Shih, T. W. (2022). Free-living marine nematodes community structure in the conservation area (Chaojing Park) and its adjacent area of Keelung, Taiwan. PLoS One, 17(5), e0268691. doi:10.1371/journal.pone.0268691
Nyaku, S. T., Sripathi, V. R., Lawrence, K., & Sharma, G. (2021). Characterizing Repeats in Two Whole-Genome Amplification Methods in the Reniform Nematode Genome. Int J Genomics, 2021, 5532885. doi:10.1155/2021/5532885
Pantó, G., Pasotti, F., Macheriotou, L., & Vanreusel, A. (2021). Combining Traditional Taxonomy and Metabarcoding: Assemblage Structure of Nematodes in the Shelf Sediments of the Eastern Antarctic Peninsula. Frontiers in Marine Science, 8. doi:10.3389/fmars.2021.629706
Pertea, G., & Pertea, M. (2020). GFF Utilities: GffRead and GffCompare. F1000Res, 9. doi:10.12688/f1000research.23297.2
Pertea, M., Pertea, G. M., Antonescu, C. M., Chang, T. C., Mendell, J. T., & Salzberg, S. L. (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol, 33(3), 290-295. doi:10.1038/nbt.3122
Piñeiro, C., Abuín, J. M., & Pichel, J. C. (2020). Very Fast Tree: speeding up the estimation of phylogenies for large alignments through parallelization and vectorization strategies. Bioinformatics, 36(17), 4658-4659. doi:10.1093/bioinformatics/btaa582
Quince, C., Lanzen, A., Davenport, R. J., & Turnbaugh, P. J. (2011). Removing noise from pyrosequenced amplicons. BMC Bioinformatics, 12, 38. doi:10.1186/1471-2105-12-38
Quinlan, A. R., & Hall, I. M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6), 841-842. doi:10.1093/bioinformatics/btq033
Ranallo-Benavidez, T. R., Jaron, K. S., & Schatz, M. C. (2020). GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nat Commun, 11(1), 1432. doi:10.1038/s41467-020-14998-3
Rhie, A., McCarthy, S. A., Fedrigo, O., Damas, J., Formenti, G., Koren, S., . . . Jarvis, E. D. (2021). Towards complete and error-free genome assemblies of all vertebrate species. Nature, 592(7856), 737-746. doi:10.1038/s41586-021-03451-0
Ridall, A., & Ingels, J. (2021). Suitability of Free-Living Marine Nematodes as Bioindicators: Status and Future Considerations. Frontiers in Marine Science, 8. doi:10.3389/fmars.2021.685327
Rödelsperger, C., Streit, A., & Sommer, R. J. Structure, Function and Evolution of The Nematode Genome. In Encyclopedia of Life Sciences.
Runnel, K., Abarenkov, K., Copot, O., Mikryukov, V., Koljalg, U., Saar, I., & Tedersoo, L. (2022). DNA barcoding of fungal specimens using PacBio long-read high-throughput sequencing. Mol Ecol Resour, 22(8), 2871-2879. doi:10.1111/1755-0998.13663
Sabina, J., & Leamon, J. H. (2015). Bias in Whole Genome Amplification: Causes and Considerations. Methods Mol Biol, 1347, 15-41. doi:10.1007/978-1-4939-2990-0_2
Sahraei, S. E., Sanchez-Garcia, M., Montoliu-Nerin, M., Manyara, D., Bergin, C., Rosendahl, S., & Rosling, A. (2022). Whole genome analyses based on single, field collected spores of the arbuscular mycorrhizal fungus Funneliformis geosporum. Mycorrhiza, 32(5-6), 361-371. doi:10.1007/s00572-022-01091-4
Santoro, A. E., Kellom, M., & Laperriere, S. M. (2019). Contributions of single-cell genomics to our understanding of planktonic marine archaea. Philos Trans R Soc Lond B Biol Sci, 374(1786), 20190096. doi:10.1098/rstb.2019.0096
Schenk, J., Kleinbolting, N., & Traunspurger, W. (2020). Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities. Ecol Evol, 10(6), 2885-2899. doi:10.1002/ece3.6104
Schenk, J., Kleinbölting, N., & Traunspurger, W. (2020). Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities. Ecology and Evolution, 10(6), 2885-2899. doi:https://doi.org/10.1002/ece3.6104
Schiffer, P. H., Kroiher, M., Kraus, C., Koutsovoulos, G. D., Kumar, S., Camps, J. I., . . . Schierenberg, E. (2013). The genome of Romanomermis culicivorax: revealing fundamental changes in the core developmental genetic toolkit in Nematoda. BMC Genomics, 14, 923. doi:10.1186/1471-2164-14-923
Sellers, G. S., Jeffares, D. C., Lawson, B., Prior, T., & Lunt, D. H. (2021). Identification of individual root-knot nematodes using low coverage long-read sequencing. PLoS One, 16(12), e0253248. doi:10.1371/journal.pone.0253248
Semprucci, F. (2013). Marine nematodes from the shallow subtidal coast of the Adriatic Sea: species list and distribution. International Journal of Biodiversity. doi:http://dx.doi.org/10.1155/2013/187659
Serra, L., Chang, D. Z., Macchietto, M., Williams, K., Murad, R., Lu, D., . . . Mortazavi, A. (2018). Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq. Bio Protoc, 8(4). doi:10.21769/BioProtoc.2729
Sidore, A. M., Lan, F., Lim, S. W., & Abate, A. R. (2016). Enhanced sequencing coverage with digital droplet multiple displacement amplification. Nucleic Acids Res, 44(7), e66. doi:10.1093/nar/gkv1493
Simao, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., & Zdobnov, E. M. (2015). BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics, 31(19), 3210-3212. doi:10.1093/bioinformatics/btv351
Sims, G. E., Jun, S. R., Wu, G. A., & Kim, S. H. (2009). Whole-genome phylogeny of mammals: evolutionary information in genic and nongenic regions. Proc Natl Acad Sci U S A, 106(40), 17077-17082. doi:10.1073/pnas.0909377106
Sivashankari, S., & Shanmughavel, P. (2007). Comparative genomics - a perspective. Bioinformation, 1(9), 376-378. doi:10.6026/97320630001376
Smit, A., Hubley, R & Green, P. (2013-2015). RepeatMasker Open-4.0. Retrieved from http://www.repeatmasker.org
Smith, M. L., Vanderpool, D., & Hahn, M. W. (2022). Using all Gene Families Vastly Expands Data Available for Phylogenomic Inference. Molecular Biology and Evolution, 39(6). doi:10.1093/molbev/msac112
Smythe, A. B., Holovachov, O., & Kocot, K. M. (2019). Improved phylogenomic sampling of free-living nematodes enhances resolution of higher-level nematode phylogeny. BMC Evol Biol, 19(1), 121. doi:10.1186/s12862-019-1444-x
Song, H., Mu, F., Sun, Y., & Hua, E. (2022). Variations of Free-Living Marine Nematode’s Taxonomic Structure and Functional Traits in Contrasting Sandy Beach Habitats. Water, 14(22), 3788. Retrieved from https://www.mdpi.com/2073-4441/14/22/3788
Spring, S., Scheuner, C., Göker, M., & Klenk, H.-P. (2015). A taxonomic framework for emerging groups of ecologically important marine gammaproteobacteria based on the reconstruction of evolutionary relationships using genome-scale data. Frontiers in Microbiology, 6. doi:10.3389/fmicb.2015.00281
Stevens, L., Martinez-Ugalde, I., King, E., Wagah, M., Absolon, D., Bancroft, R., . . . Blaxter, M. (2023). Ancient diversity in host-parasite interaction genes in a model parasitic nematode. bioRxiv, 2023.2004.2017.535870. doi:10.1101/2023.04.17.535870
Subbotin, S. A., Oliveira, C. J., Alvarez-Ortega, S., Desaeger, J. A., Crow, W., Overstreet, C., . . . Inserra, R. N. (2021). The taxonomic status of Aphelenchoides besseyi Christie, 1942 (Nematoda: Aphelenchoididae) populations from the southeastern USA, and description of Aphelenchoides pseudobesseyi sp. n. Nematology, 23(4), 381-413. doi:10.1163/15685411-bja10048
Szitenberg, A., Cha, S., Opperman, C. H., Bird, D. M., Blaxter, M. L., & Lunt, D. H. (2016). Genetic Drift, Not Life History or RNAi, Determine Long-Term Evolution of Transposable Elements. Genome Biol Evol, 8(9), 2964-2978. doi:10.1093/gbe/evw208
Teramoto, M., Yagyu, K.-i., & Nishijima, M. (2015). Perspicuibacter marinus gen. nov., sp. nov., a semi-transparent bacterium isolated from surface seawater, and description of Arenicellaceae fam. nov. and Arenicellales ord. nov. International Journal of Systematic and Evolutionary Microbiology, 65(Pt_2), 353-358. doi:https://doi.org/10.1099/ijs.0.064683-0
Tsai, I. J., Hunt, M., Holroyd, N., Huckvale, T., Berriman, M., & Kikuchi, T. (2014). Summarizing specific profiles in Illumina sequencing from whole-genome amplified DNA. DNA Res, 21(3), 243-254. doi:10.1093/dnares/dst054
Tyson, J. R., O'Neil, N. J., Jain, M., Olsen, H. E., Hieter, P., & Snutch, T. P. (2018). MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome. Genome Res, 28(2), 266-274. doi:10.1101/gr.221184.117
Van Hien, H., Thi Dung, B., Ngo, H. D., & Doanh, P. N. (2021). First morphological and molecular identification of third-stage larvae of Anisakis typica (Nematoda: Anisakidae) from marine fishes in Vietnamese water. J Nematol, 53. doi:10.21307/jofnem-2021-010
Vaser, R., Sovic, I., Nagarajan, N., & Sikic, M. (2017). Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res, 27(5), 737-746. doi:10.1101/gr.214270.116
Vieira, S., Sroczyńska, K., Neves, J., Martins, M., Costa, M. H., Adão, H., & Vicente, C. S. L. (2023). Distribution patterns of benthic bacteria and nematode communities in estuarine sediments. Estuarine, Coastal and Shelf Science, 291, 108448. doi:https://doi.org/10.1016/j.ecss.2023.108448
Viney, M. (2017). How Can We Understand the Genomic Basis of Nematode Parasitism? Trends Parasitol, 33(6), 444-452. doi:10.1016/j.pt.2017.01.014
Wang, J., Gao, S., Mostovoy, Y., Kang, Y., Zagoskin, M., Sun, Y., . . . Davis, R. E. (2017). Comparative genome analysis of programmed DNA elimination in nematodes. Genome Res, 27(12), 2001-2014. doi:10.1101/gr.225730.117
Warris, S., Schijlen, E., van de Geest, H., Vegesna, R., Hesselink, T., Te Lintel Hekkert, B., . . . de Ridder, D. (2018). Correcting palindromes in long reads after whole-genome amplification. BMC Genomics, 19(1), 798. doi:10.1186/s12864-018-5164-1
Weinstein, D. J., Allen, S. E., Lau, M. C. Y., Erasmus, M., Asalone, K. C., Walters-Conte, K., . . . Bracht, J. R. (2019). The genome of a subterrestrial nematode reveals adaptations to heat. Nat Commun, 10(1), 5268. doi:10.1038/s41467-019-13245-8
Wood, D. E., Lu, J., & Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biol, 20(1), 257. doi:10.1186/s13059-019-1891-0
Wu, T. C., Lei, W. Y., Chen, M. C., & Hu, C. T. (2012). Strongyloides stercoralis infection: a health issue regarding indigenous people in Taiwan. Trans R Soc Trop Med Hyg, 106(8), 468-472. doi:10.1016/j.trstmh.2012.04.004
Xie, Y., Zhang, P., Xue, B., Cao, X., Ren, X., Wang, L., . . . Zhang, L. (2020). Establishment of a marine nematode model for animal functional genomics, environmental adaptation and developmental evolution. bioRxiv, 2020.2003.2006.980219. doi:10.1101/2020.03.06.980219
Yokono, M., Satoh, S., & Tanaka, A. (2018). Comparative analyses of whole-genome protein sequences from multiple organisms. Sci Rep, 8(1), 6800. doi:10.1038/s41598-018-25090-8
Yoshimura, J., Ichikawa, K., Shoura, M. J., Artiles, K. L., Gabdank, I., Wahba, L., . . . Schwarz, E. M. (2019). Recompleting the Caenorhabditis elegans genome. Genome Res, 29(6), 1009-1022. doi:10.1101/gr.244830.118
Zhang, C., Scornavacca, C., Molloy, E. K., & Mirarab, S. (2020). ASTRAL-Pro: Quartet-Based Species-Tree Inference despite Paralogy. Mol Biol Evol, 37(11), 3292-3307. doi:10.1093/molbev/msaa139
Zhu, Q., Mai, U., Pfeiffer, W., Janssen, S., Asnicar, F., Sanders, J. G., . . . Knight, R. (2019). Phylogenomics of 10,575 genomes reveals evolutionary proximity between domains Bacteria and Archaea. Nat Commun, 10(1), 5477. doi:10.1038/s41467-019-13443-4
Zou, H., Chen, F. L., Li, W. X., Li, M., Lei, H. P., Zhang, D., . . . Wang, G. T. (2022). Inverted base composition skews and discontinuous mitochondrial genome architecture evolution in the Enoplea (Nematoda). BMC Genomics, 23(1), 376. doi:10.1186/s12864-022-08607-4