Abstract A26: Comparison of DNA extraction kits for metagenomic studies in feces

Abstract

Abstract DNA extraction is a critical step in metagenomic studies since it requires the proper lysis of cell walls, allowing the recovery of nucleic acids that would represent the total diversity of communities. Recent studies indicated that the use of different DNA extraction methods in the same sample yielded distinct microbial profiles, highlighting the importance of standardization in DNA extraction methodologies to accurately measure the human microbiome. These issues are even more critical when limiting amounts of samples are available, or when studies target communities with reduced abundance, such as fungi, which represents only 0.1% of total gut microbiota, as has been poorly explored. Here we investigated the performance of three commercial kits for DNA extraction from human fecal samples: QIAamp DNA Stool Mini Kit (Qiagen), E.Z.N.A. Stool DNA Kit (Omega), and ZymoBIOMICS DNA Kit (Zymo), all with or without adding an enzymatic extraction with MetaPolyzyme (MAC4L - Sigma), in an attempt to improve the microbiome community profiling. After extraction, DNA-quality and yield were evaluated using Qubit and real-time PCR, V4-V5 16S rRNA and ITS2 followed by DNA sequencing, as well as further analysis using shotgun DNA sequencing. No significant differences were observed in DNA yield as evaluated by Qubit; an average of 40.3 ng/ul of DNA was obtained for all samples and kits. For both fungi and bacteria analysis, the addition of MAC4L in the extraction protocol apparently did not change the alpha diversity (observed and Shannon) for both individuals. Our analysis for samples extracted with different kits, with or without MAC4L, indicated the percentage of reads from bacteria to vary between 85-90%, whereas fungi were between 0.29-0.49%. Human DNA was also found, as expected, and relative amounts varied from 0.1 to 0.3%. qPCR showed that for all kits up to 50ng of DNA/15uL reaction can be safely used, with no significant DNA polymerase inhibition. These results showed similar efficacy for the three kits in representing bacteria and fungi from feces, and maybe because all these kits had a bead-beading step we had no clear benefits from adding extra enzymatic digestion steps with MAC4L. Finally, we decided to use the Omega kit in our lab, since it combines a simple extraction protocol and is reliable and cost-effective. Citation Format: Isabella Kuniko T. Takenaka, Bruno S. Moda, Thaís F. Bartelli, Maria G. Amorim, Gabriela E. Albuquerque, Andrew M. Thomas, Emmanuel Dias-Neto, Diana N. Nunes. Comparison of DNA extraction kits for metagenomic studies in feces [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A26.