High-throughput sequencing of nematode communities from total soil DNA extractions

Annals of Occupational and Environmental Medicine

Rumakanta Sapkota¹  Mogens Nicolaisen¹ 

[1] Department of Agroecology, Aarhus University, Faculty of Science and Technology, Forsøgsvej 1, Slagelse, DK-4200, Denmark

关键词: rDNA;    18S;    Diversity;    SSU;    Next-generation sequencing;    Community;    Nematode;   

Others  :  1128391
DOI  :  10.1186/s12898-014-0034-4

 received in 2014-05-13, accepted in 2014-12-18,  发布年份 2015

【 摘 要 】

Background

Nematodes are extremely diverse and numbers of species are predicted to be more than a million. Studies on nematode diversity are difficult and laborious using classical methods and therefore high-throughput sequencing is an attractive alternative. Primers that have been used in previous sequence-based studies are not nematode specific but also amplify other groups of organisms such as fungi and plantae, and thus require a nematode enrichment step that may introduce biases.

Results

In this study an amplification strategy which selectively amplifies a fragment of the SSU from nematodes without the need for enrichment was developed. Using this strategy on DNA templates from a set of 22 agricultural soils, we obtained 64.4% sequences of nematode origin in total, whereas the remaining sequences were almost entirely from other metazoans. The nematode sequences were derived from a broad taxonomic range and most sequences were from nematode taxa that have previously been found to be abundant in soil such as Tylenchida, Rhabditida, Dorylaimida, Triplonchida and Araeolaimida.

Conclusions

Our amplification and sequencing strategy for assessing nematode diversity was able to collect a broad diversity without prior nematode enrichment and thus the method will be highly valuable in ecological studies of nematodes.

【 授权许可】

   
2015 Sapkota and Nicolaisen; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Lambshead PJD. Marine nematode biodiversity. In: Nematology: Advances and Perspectives, Vol 1 Edited by Chen ZX. Chen, SY, Dickson DW: CABI Publishing; 2004. p. 438–68.
• [2]Yeates GW, Bongers T, Degoede RGM, Freckman DW, Georgieva SS: Feeding-habits in soil nematode families and genera – an outline for soil ecologists. J Nematol. 1993, 25:315-31.
• [3]Agrios GN: Plant Pathology. 5th edition. Academic Press, London, UK; 2005.
• [4]Holterman M, Karssen G, van der Elsen S, van Megen H, Bakker J, Helder J: Small subunit rDNA-based phylogeny of the Tylenchida sheds light on relationships among some high-impact plant-parasitic nematodes and the evolution of plant feeding. Phytopathology. 2009, 99:227-35.
• [5]Jones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MGK, et al.: Top 10 plant -parasitic nematodes in molecular plant pathology. Mol Plant Pathol. 2013, 14:946-61.
• [6]Zhao J, Neher DA: Soil nematode genera that predict specific types of disturbance. Appl Soil Ecol. 2013, 64:135-41.
• [7]Porazinska DL, Giblin-Davis RM, Faller L, Farmerie W, Kanzaki N, Morris K, et al.: Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversity. Mol Ecol Resour. 2009, 9:1439-50.
• [8]Porazinska DL, Sung W, Giblin-Davis RM, Kelley TW: Reproducibility of read numbers in high-throughput sequencing analysis of nematode community composition and structure. Mol Ecol Resour. 2010, 10:666-76.
• [9]Morise H, Miyazaki E, Yoshimitsu S, Eki T: Profiling nematode communities in unmanaged flowerbed and agricultural field soils in Japan by DNA barcode sequencing. PLoS One. 2012, 7:e51785.
• [10]Darby BJ, Todd TC, Herman MA: High-throughput amplicon sequencing of rRNA genes requires a copy number correction to accurately reflect the effects of management practices on soil nematode community structure. Mol Ecol. 2013, 22:5456-71.
• [11]Porazinska DL, Giblin-Davis RM, Powers TO, Kelley TW: Nematode spatial and ecological patterns from tropical and temperate rainforests. PLoS One. 2012, 7:e44641.
• [12]Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al.: The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013, 41:D590-6.
• [13]Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, et al.: A molecular evolutionary framework for the phylum Nematoda. Nature. 1998, 392:71-5.
• [14]Viglierchio DR, Schmitt RV: On the methodology of nematode extraction from field samples: Baermann funnel modifications. J Nematol. 1983, 15:438-44.
• [15]Creer S, Fonseca VG, Porazinska DL, Giblin-Davis RM, Sung W, Power DM, et al.: Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises. Mol Ecol. 2010, 19:4-20.
• [16]Briar SS, Fonte SJ, Park I, Six J, Scow K, Ferris H: The distribution of nematodes and soil microbial communities across soil aggregate fractions and farm management systems. Soil Biol Biochem. 2011, 43:905-14.
• [17]Li Y, Cao Z, Hu C, Li J, Yang H: Response of nematodes to agricultural input levels in various reclaimed and unclaimed habitats. Eur J Soil Biol. 2014, 60:120-9.
• [18]Neher DA: Nematode communities in organically and conventionally managed agricultural soils. J Nematol. 1999, 31:142-54.
• [19]Yeates GW, Newton PCD: Long-term changes in topsoil nematode populations in grazed pasture under elevated atmospheric carbon dioxide. Biol Fertil Soils. 2009, 45:799-808.
• [20]Amend AS, Seifert KA, Bruns TD: Quantifying microbial communities with 454 pyrosequencing: does read abundance count? Mol Ecol. 2010, 19:5555-65.
• [21]van Megen H, van den Elsen S, Holterman M, Karssen G, Mooyman P, Bongers T, et al.: A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences. Nematology. 2009, 11:927-50.
• [22]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28:2731-9.
• [23]Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al.: QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010, 7:335-6.
• [24]Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ: Removing noise from pyrosequenced amplicons. BMC Bioinformatics. 2011, 12:239. BioMed Central Full Text
• [25]Sapkota, R, Nicolaisen, M. Nematode sequence data from 22 soil samples. 2014. doi:10.5061/dryad.0h653