Comparison of DNA Extraction Methods for Metagenomic Sequencing in Anaerobic Digestion

Abstract

Objectives:This study was performed to compare three commercial kits for the extraction of genomic DNA from anaerobic digestate for subsequent iSeq 100 sequencing and microbial community analysis.Methods:A full-scale mesophilic biogas plant was sampled, and made into aliquots of identical volumes to extract DNA using three commercial kits: FastDNA spin kit for soil (MP Biomedicals, USA), Exgene stool DNA mini (GeneAll, South Korea) and AccuPrep genomic DNA extraction kit (Bioneer, South Korea). To analyze the microbial communities in the purified DNA, 16S rDNA amplicon sequencing (V3-4 region for bacteria and V4-5 region for archaea) was performed on the Illumina iSeq 100 platform. Quality filtered sequence reads were OTU-clustered for taxonomic assignment conducted using the RDP classifier on-line.Results and Discussion:The microbial community structure visualized on the NMDS plot using the weighted UniFrac distance revealed that both bacteria and archaeal communities have phylogenetic differences depending on the DNA extraction kit used. In addition, the abundance of certain microbial populations was significantly different among the DNA extraction methods. For example, Proteobacteria was the least abundant using the soil kit, while this phylum was the most abundant when the stool kit was used. However, in the case of Thermotogae, this tendency was vice versa. The abundance of archaeal genera Methanomethylovorans and Methanosarcina was also affected by DNA extraction methods.Conclusions:The microbial populations analyzed by 16S based sequencing were affected by DNA extraction methods. To compare microbial community changes in the identical set of research, one DNA extraction method should be chosen and used consistently for the whole experiment.