Effects of different DNA extraction methods on genomic DNA extraction and amplicon libraries of Daqu.

Daqu is a typical multi-enzyme and multi-microbes fermentation starter of traditional Chinese liquor. The culture-independent molecular method has become a general method for the study of the Daqu microbial community. To determine the most suitable DNA extraction method of Daqu samples, the effects of different DNA extraction methods on Daqu genomic DNA extraction and amplicon libraries were evaluated. In this study, four DNA extraction methods were selected for the extraction of microbial DNA from two kinds of Daqu. Combined with DNA extraction quality, qPCR quantitative results, Illumina Miseq sequencing results of bacterial 16S rRNA gene, and fungal ITS gene amplicon library, the effects of different extraction methods of Daqu genomic DNA were evaluated. The comprehensive analysis of DNA yield and microbial biomass showed that the DNA yield of the SDS-based extraction method was higher than that of the kit extraction method, and the DNA yield of the in-situ SDS-based extraction method was as high as 5.46 x 10⁴ ng/g, but the DNA purity was low. The qPCR quantitative results of the SDS-based extraction method were 101 - 6 orders of magnitude higher than those of the kit extraction method, and the microbial biomass of the washing-pretreatment plus SDS-based extraction method was the highest, which was 10^1-3 orders of magnitude higher than that of the in-situ SDS-based extraction method. Whereas the microbial biomass of the DNA extraction kit was the lowest, with only 10^2-4of the Shannon diversity index among amplicon libraries showed that different DNA extraction methods had significant differences among fungal ITS amplicon libraries, but no significant differences among bacterial 16S rDNA amplicon libraries. The Shannon index of the fungal amplicon library of the washing-pretreated samples were higher than that of the in-situ treated samples. By comparing the operational taxonomic units (OTU) with significant differences among the amplicon libraries, it could be seen that most of the OTUs in the bacterial 16S rDNA amplicon library had differences in abundance, but most of them could be detected in different DNA extraction methods. However, among the fungal ITS amplicon libraries, most OTUs showed a similar trend between different DNA extraction methods, indicating that the DNA extraction method had a significant effect on the extraction of some fungal microbial genomes, and the washing-pretreatment was beneficial to the extraction of fungal genomic DNA. Compared with the four DNA extraction methods, the washing-pretreatment plus SDS-based extraction method was the most suitable for the quantitative and community structure analysis of Daqu microorganisms. [ABSTRACT FROM AUTHOR]