Effect of DNA isolation method and host species on the metagenomic DNA recovery and microbial community coverage

The introduction of next generation sequencing to characterize rumen microbial diversity has expanded the scope of animal nutrition to understand ruminal fermentation in greater details. Although diis technique is highly accurate, it suffers from biases introduced at different stages of sample processing, sequencing and bioinformatics analysis. It is well established that the outcome of metagenomics (using DNA) is affected by the source and method of DNA isolation. The present study aimed to evaluate two popular rumen metagenomic DNA isolation methods, Repeated Bead Beating plus Column (RBBC) and ZR Fecal DNA Microprep Kit, for their effect on the DNA quality and quantity, and microbial community assessment in four ruminant species, namely cattle, buffalo, goat and sheep. Metagenomic DNA was isolated from total rumen contents using these methods, and the quality and quantity of the purified DNA were determined. The coverage of microbial diversity was assessed by the Terminal Restriction Fragment Length Polymorphism (TRFLP) technique using two restriction enzymes (Mspl and BsuRI). The number and peak area of terminal restriction fragments (TRFs) were used to compare the efficiency of the DNA isolation methods across the species. The study established that although the metagenomic DNA recovery was greater using the RBBC method, the quality of the purified DNA was superior with die ZR Fecal DNA kit. Significantly greater microbial diversity (P<0.05) was also noticed in cattle, goat and sheep rumen metagenomic DNA isolated using the ZR Fecal DNA kit. Further, the overall comparison between the methods, irrespective of the species, also revealed significantly (P< 0.001) greater microbial diversity coverage in the ZR Fecal DNA isolation kit than the RBBC method. In conclusion, the current study revealed that die ZR Fecal DNA Kit was comparatively better than the RBBC method for assessing microbial diversity in me rumen metagenomic samples of cattle, buffalo, goat and sheep.