Home >Current Issue

Volume: 7, Issue: 1, Jan-Feb, 2019
DOI: 10.7324/JABB.2019.70103

Research Article

Comparative analysis of metagenomic DNA extraction methods from gut microbiota of zebrafish (Danio rerio) for downstream next-generation sequencing


Venetia D’Rose, Tina Kollannoor Johny, Sarita Bhat

  Author Affiliations


Abstract

Zebrafish (Danio rerio) being a well-established model system can provide significant understanding about interactions between gut microbiota and host system. Gut bacterial diversity may be unraveled only by analyzing metagenomic DNA from diverse higher organisms which helps to identify gut microbes beneficial to the host. Isolation and standardization of metagenomic DNA from zebrafish gut are an inconsequential, but demanding process, as most microbes in the gastrointestinal tract are unculturable. A variety of commercial kits and modified protocols is used for bacterial metagenomic DNA extraction. However, only few methods are reported for the effective isolation of community DNA from zebrafish. The present work portrays an augmented method for gut metagenomic DNA isolation from zebrafish gut, focused on obtaining effective yield and purity. Three methods using kit were evaluated, of which the modified kit method was an affordable and feasible method for isolation of metagenomic DNA from zebrafish. The modified protocol can be used for isolating quality DNA which is an important parameter for downstream applications such as polymerase chain reaction, cloning, next-generation sequencing, and others.

Keywords:

DNA isolation, Gut microbiota, Metagenomics, Next-generation sequencing, Zebrafish.



Citation: D’Rose V, Johny TK, Bhat S. Comparative analysis of metagenomic DNA extraction methods from gut microbiota of zebrafish (Danio rerio) for downstream next-generation sequencing. J App Biol Biotech. 2019;7(01):11-15. DOI: 10.7324/JABB.2019.70103


Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

1. Hooper, L. V., Wong, M. H., Thelin, A., Hansson, L., Falk, P. G., & Gordon, J. I. (2001). Molecular analysis of commensal host-microbial relationships in the intestine. Science, 291(5505), 881-884. https://doi.org/10.1126/science.291.5505.881

2. Nayak, S.K. (2010). Role of gastrointestinal microbiota in fish, Aquac. Res. 41 1553e1573.

3. Romero, J., E. Ringø, D.L. Merrifield (2014). The gut microbiota of fish, in Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics, John Wiley & Sons, Ltd., West Sussex, 2014, pp. 75e100

4. Ringø, E., R.E. Olsen, J.L.G. Vecino, S. Wadsworth, S.K. Song (2012). Use of immunostimulants and nucleotides in aquaculture: a review, J. Mar. Sci. Res. Dev. 2.

5. Silva, F. C. D. P., Nicoli, J. R., Zambonino-Infante, J. L., Kaushik, S., &Gatesoupe, F. J. (2011). Influence of the diet on the microbial diversity of faecal and gastrointestinal contents in gilthead sea bream (Sparusaurata) and intestinal contents in goldfish (Carassiusauratus). FEMS microbiology ecology, 78(2), 285-296. https://doi.org/10.1111/j.1574-6941.2011.01155.x

6. Carda-Dieguez, M., A. Mira, B. Fouz (2014). Pyrosequencing survey of intestinal microbiota diversity in cultured sea bass (Dicentrarchuslabrax) fed functional diets, FEMS Microbiol. Ecol. 87, 451e459.

7. Ravin, N. V., Mardanov, A. V., &Skryabin, K. G. (2015). Metagenomics as a tool for the investigation of uncultured microorganisms. Russian Journal of Genetics, 51(5), 431-439. https://doi.org/10.1134/S1022795415050063

8. Andersson, A.F., M. Lindberg, H. Jakobsson, F. Backhed, P. Nyre N, L. Engstrand (2008). Comparative analysis of human gut microbiota by barcoded pyrosequencing, PLoS One 3 e2836. https://doi.org/10.1371/journal.pone.0002836

9. Zhang, C., M. Zhang, S. Wang, R. Han, Y. Cao, W. Hua, Y. Mao, X. Zhang, X. Pang, C. Wei, G. Zhao, Y. Chen, L. Zhao (2010).Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice, ISME J. 4 (2010) 232e241.

10. J.F. Garcia-mazcorro, D.J. Lanerie, S.E. Dowd, C.G. Paddock, N. Gr\ützner, J.M. Steiner, R. Ivanek, J.S. Suchodolski (2011). Effect of a multi-species synbiotic formulation on fecal bacterial microbiota of healthy cats and dogs as evaluated by pyrosequencing, FEMS Microbiol. Ecol. 78, 542e554. https://doi.org/10.1111/j.1574-6941.2011.01185.x

11. Hart, M. L., Meyer, A., Johnson, P. J., & Ericsson, A. C. (2015). Comparative evaluation of DNA extraction methods from feces of multiple host species for downstream next-generation sequencing. PloS one, 10(11), e0143334. https://doi.org/10.1371/journal.pone.0143334

12. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor.

13. Shivaji S., Bhanu N. V., Aggarwal R. K. (2000). Identification of Yersinia pestisas the causative organism of plague in India as determined by 16S rDNA sequencing and RAPD-based genomic fingerprinting. FEMS Microbiology Letters 189 (2): 247-252. https://doi.org/10.1111/j.1574-6968.2000.tb09238.x

14. Larsen, A. M., Mohammed, H. H., & Arias, C. R. (2014). Comparison of DNA extraction protocols for the analysis of gut microbiota in fishes. FEMS microbiology letters, 362(5). https://doi.org/10.1093/femsle/fnu031

15. Lantz PG, Matsson M, Wadstrom T (1997). Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J Microbiol Meth 1997; 28:159–67. https://doi.org/10.1016/S0167-7012(97)00979-2

16. Walker, A.W., Martin, J.C., Scott, P., Parkhill, J., Flint, H.J. and Scott, K.P. (2015) 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. Microbiome 3, 26. https://doi.org/10.1186/s40168-015-0087-4

17. Ringø, E., Zhou, Z., Vecino, J. G., Wadsworth, S., Romero, J., Krogdahl, Å. & Owen, M. (2016). Effect of dietary components on the gut microbiota of aquatic animals. A never‐ending story? Aquaculture Nutrition, 22(2), 219-282. https://doi.org/10.1111/anu.12346

18. Roeselers, G., Mittge, E. K., Stephens, W. Z., Parichy, D. M., Cavanaugh, C. M., Guillemin, K., & Rawls, J. F. (2011). Evidence for a core gut microbiota in the zebrafish. The ISME journal, 5(10), 1595. https://doi.org/10.1038/ismej.2011.38

19. Borrelli, L., Aceto, S., Agnisola, C., De Paolo, S., Dipineto, L., Stilling, R. M., &Fioretti, A. (2016). Probiotic modulation of the microbiota-gut-brain axis and behaviour in zebrafish. Scientific reports, 6, srep30046. https://doi.org/10.1038/srep30046

20. Udayangani, R. M. C., Dananjaya, S. H. S., Nikapitiya, C., Heo, G. J., Lee, J., & De Zoysa, M. (2017). Metagenomics analysis of gut microbiota and immune modulation in zebrafish (Danio rerio) fed chitosan silver nanocomposites. Fish & shellfish immunology, 66, 173-184. https://doi.org/10.1016/j.fsi.2017.05.018

21. Gallagher, S. R., & Desjardins, P. R. (2007). Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current protocols in human genetics, A-3D. https://doi.org/10.1002/0471142905.hga03ds53

22. Martin-Laurent, F., Philippot, L., Hallet, S., Chaussod, R., Germon, J. C., Soulas, G., &Catroux, G. (2001). DNA extraction from soils: old bias for new microbial diversity analysis methods. Applied and environmental microbiology, 67(5), 2354-2359. https://doi.org/10.1128/AEM.67.5.2354-2359.2001

23. Lakay, F. M., Botha, A., & Prior, B. A. (2007). Comparative analysis of environmental DNA extraction and purification methods from different humic acid‐rich soils. Journal of applied microbiology, 102(1), 265-273. https://doi.org/10.1111/j.1365-2672.2006.03052.x

24. Guti\érrez-Lucas, L. R., Montor-Antonio, J. J., Cort\és-L\ópez, N. G., &del Moral, S. (2014). Strategies for the extraction, purification and amplification of metagenomic DNA from soil growing sugarcane. Advances in Biological Chemistry, 4(04), 281. https://doi.org/10.4236/abc.2014.44034

25. Teske, A., &Sørensen, K. B. (2008). Uncultured archaea in deep marine subsurface sediments: have we caught them all? The ISME journal, 2(1), 3. https://doi.org/10.1038/ismej.2007.90

26. Patel, J. B. (2001). 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Molecular Diagnosis, 6(4), 313-321. https://doi.org/10.2165/00066982-200106040-00012

Article Metrics

Similar Articles