Background and Aim: A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.

Materials and Methods: Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.

Results: Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme- Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/μl and 161 ng/μl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A_260nm/A_280nm) and 2.3 (A_260nm/A_230nm) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at -80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as Prevotella ruminicola, Streptococcus lutetiensis, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Selenomonas ruminantium.

Conclusion: To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment.

Keywords: 16S rRNA gene sequencing, community DNA extraction, goat, polymerase chain reaction, rumen digesta.

1. Griffiths, R.J., Whiteley, A.S., O'Donell, A.G. and Bailey, M.J. (2000) Rapid method for co extraction of DNA and RNA from natural environment for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl. Environ. Microbiol, 66(12): 5488-5491. [Crossref]

2. Henderson, G., Cox, F., Kittelmann, S., Miri, V.H., Zethof, M., Noel, S.J., Waghorn, G.C. and Janssen, P.H. (2013) Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities. PLoS One, 8(9): 1-14. [Crossref]

3. Henderson, G., Cox, F., Ganesh, S., Jonker, A., Young, W., Global Rumen Census Collaborators. and Janssen, P.H. (2015) Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. Sci. Rep-UK., 5: 14567. [Crossref]

4. Klieve, A.V., O Leary, M.N., Mc Millen, L. and Ouwerkerk, D. (2007) Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet. J. Appl. Microbiol., 103(6): 2065-2073. [Crossref] [PubMed]

5. McSweeney, C.S., Denman, S.E., Wright, A.D.G. and Yu, Z. (2007) Application of recent DNA/RNA-based techniques in rumen ecology. Asian-Aust. J. Anim. Sci., 20(2): 283-294. [Crossref]

6. Rosero, J.A., Strosova, L., Mrazek, J., Fliegerova, K. and Kopecny, J. (2012) PCR detection of uncultured rumen bacteria. Folia Microbiol., 57: 325-330. [Crossref] [PubMed]

7. Kang, T.J. and Yang, M.S. (2004) Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants. BMC Biotechnol., 4: 20. [Crossref] [PubMed] [PMC]

8. Vaidya, J.D., Bogertm, B.V.D., Edwards, J.E., Boekhorst, J., Gastelen, S.V., Saccenti, E., Plugge, C.M. and Smidt, H. (2018) The effect of DNA extraction methods on observed microbial communities from fibrous and liquid rumen fractions of dairy cows. Front Microbiol., 9: 1-16. [Crossref] [PubMed] [PMC]

9. Tatiana, Y., Salcedo, G., Ramirez-Uscategui, R.A., Machado, E.G., Messana, J.D., Kishi, L.T., Dias, A.V.L. and Berchielli, T.T. (2017) Studies on bacterial community composition are affected by the time and storage method of the rumen content. PLOS One, 12(4), 1-15.

10. Stiverson, J., Morrison, M. and Yu, Z. (2011) Populations of select cultured and uncultured bacteria in the Rumen of sheep and the effect of diets and Ruminal fractions. Int. J. Microbiol., 2011: 1-8. [Crossref]

11. Tajima, K., Aminov, R.I., Nagamine, T., Matsui, H., Nakamura, M. and Benno, Y. (2001) Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl. Environ. Microbiol 67(6): 2766-2774. [Crossref] [PubMed] [PMC]

12. Lever, M.A., Torti, A., Eickenbusch, P., Michaud, A.B., Santl-Temkiv, T. and Jorgensen, B.B. (2015) A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types. Methods, 6: 476. [Crossref]

13. Jami, E., Israel, A., Kotser, A. and Mizrahi, I. (2009) Exploring the bovine rumen bacterial community from birth to adulthood. The ISME J, 7: 1069-1079. [Crossref] [PubMed] [PMC]

14. Jin, W., Cheng, Y. and Zhu, W. (2017) The community structure of Methanomassiliicoccales in the rumen of Chinese goats and its response to a high grain diet. J Anim. Sci. Biotech., 8: 47. [Crossref] [PubMed] [PMC]

15. Minas, K., McEwan, N.R., Jamie, C. and Scott, N.K.P. (2011) Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures. FEMS Microbiol. Lett., 325: 162-169. [Crossref]

16. Popova, M., Martin, C. and Morgavi, D.P. (2010) Improved protocol for high-quality Co-extraction of DNA and RNA from rumen digesta. Folia Microbiol., 55(4): 368-372. [Crossref] [PubMed]

17. Bashir, Y., Rather, I.A. and Konwar, B.K. (2015) Rapid and simple DNA extraction protocol from goat rumen digesta for metagenomic analysis. Pak. J. Pharm. Sci., 28(6): 2305-2309. [PubMed]

18. Lead, J.R., Starchev, K. and Wilkinson, K.J. (2003) Diffusion coefficients of humic substances in agarose gel and water. Environ. Sci. Technol., 37: 482-487. [Crossref] [PubMed]

19. Chen, Y.B., Lan, D.L., Tang, C., Yang, X.N. and Jian, L. (2015) Effect of DNA extraction methods on the apparent structure of yak rumen microbial communities as revealed by 16S rDNA sequencing. Pol. J Microbiol., 64(1): 29-36. [PubMed]

20. Yu, Z. and Morrison, M. (2004) Improved extraction of PCR-quality community DNA from digesta and fecal samples. Bio Tech., 36: 808-812. [Crossref]

21. Fliegerova, K., Tapio, I., Bonin, A., Mrazek, J., Callegaric, M.L., Bani, P., Bayat, A., Vilkki, J., Kope_cny, J., Shingfieldd, K.J., Boyer, F., Coissac, E., Taberlet, P. and Wallace, R.J. (2014) Effect of DNA extraction and sample preservation method on rumen bacterial population. Anaerobe, 29: 80-84. [Crossref] [PubMed]

22. Andersen, A.W., Bahl, M.I., Carvalho, V., Kristiansen, K., Sicheritz-Ponten, T., Gupta, R. and Licht, T.R. (2014) Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis. Microbiome, 2(19): 1-11.

23. Zhao, F. and Xua, K. (2012) Efficiency of DNA extraction methods on the evaluation of soil micro-eukaryotic diversity. Acta Ecol. Sinica, 32: 209-214. [Crossref]

24. Ross, E.M., Moate, P.J., Bath, C.R., Davidson, S.R., Sawbridge, T.I., Guthridge, K.M., Cocks, B.G. and Hayes, B.J. (2012) High throughput whole rumen metagenome profiling using untargeted massively parallel sequencing. BMC Genet., 13(53): 1-14. [Crossref]

25. Nettmann, E., Bergmann, I., Mundt, K., Linke, B. and Klocke, M. (2008) Archaea diversity within a commercial biogas plant utilizing herbal biomass determined by 16S rDNA and mcrA analysis. J. Appl. Microbiol., 105: 1835-1850. [Crossref] [PubMed]

26. Liu, J.H., Zhang, M., Zhang, R., Zhu, W. and Mao, S. (2016) Comparative studies of the composition of bacterial microbiota associated with the ruminal content, ruminal epithelium and in the faeces of lactating dairy cows. Microbial. Biotech., 9(2): 257-268. [Crossref] [PubMed] [PMC]

27. Kang, S., Denman, S.E., Morrison, M., Yu, Z. and McSweeney, C.S. (2009) An efficient RNA extraction method for estimating gut microbial diversity by polymerase chain reaction. Curr. Microbiol., 58(5): 464-471. [Crossref] [PubMed]

28. Pers-Kamczyc, E., Zmora, P., Cieslak, A. and Szumacher-Strabel, M. (2011) Development of nucleic acid-based techniques and possibilities of their application to rumen microbial ecology research. J Anim. Feed Sci., 20: 315-337. [Crossref]

29. Li, M., Penner, G.B., Hernandez-Sanabria, E., Oba, M. and Guan, L.L. (2009) Effects of sampling location and time, and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen. J. Appl. Microbiol., 107: 1924-1934. [Crossref]

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