Microbiome analyses of poultry feeds: Part I. Comparison of five different DNA extraction methods

Authors: OLSON, ELENA - University Of Wisconsin; DITTOE, DANA - University Of Wisconsin; MICCICHE, ANDREW - University Of Arkansas; STOCK, DAVID - Stetson University; RUBINELLI, PETER - University Of Arkansas; Rothrock, Michael; RICKE, STEVEN - University Of Wisconsin

Submitted to: Journal of Environmental Science and Health
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2023
Publication Date: 5/23/2024
Citation: Olson, E.G., Dittoe, D.K., Micciche, A.C., Stock, D.A., Rubinelli, P.M., Rothrock Jr, M.J., Ricke, S.C. 2024. Microbiome analyses of poultry feeds: Part I. Comparison of five different DNA extraction methods. Journal of Environmental Science and Health. https://doi.org/10.1080/03601234.2024.2353002.
DOI: https://doi.org/10.1080/03601234.2024.2353002

Interpretive Summary: There is no particular DNA extraction kit available for animal feed due to the wide range of variability in feed composition. An optimized DNA extraction technique associated with feed is necessary for comparable downstream sequencing analyses. In this two-part study, the variation in microbiota of five DNA extraction techniques (Part I ) and the microbial compositional diversities of nine commercial poultry feeds (Part II ) were compared. DNA extraction methods included Qiagen QIAamp DNA Stool Mini Kit (Qiagen), modified Qiagen with Lysing Matrix B (MQ), modified Qiagen with celite purification (MQC), polyethylene glycol (PEG), and 1-Day Direct. Genomic DNA was amplified using custom dual-indexed primers and sequenced on an Illumina MiSeq. Sequencing data were analyzed in QIIME2-2021.4 with alpha and beta diversity being determined using Kruskal-Wallis and ANOSIM. Compositional taxonomic differences were determined using analysis of composition of microbiomes (ANCOM). Main and pairwise effects were considered significant at P = 0.05 and Q = 0.05. Results of Part I suggest that there is a direct impact on the diversity and compositional differences based on the extraction method used. Extraction of feed with the Qiagen method produced the least evenness and richness compared to the other protocols used, MQ, MQC, PEG, and 1-Day Direct (P < 0.05). Bead beating and lysozyme application were used in the 1-Day Direct and PEG protocols which produced greater bacterial diversities (P < 0.05). PEG and 1-Day Direct produced numerically greater relative abundance of different genera and phyla associated with extraction methods. Although the use of the Qiagen kit did not result in an increased compositional diversity, application of these kits with adjustments, such as MQ and MQC, can provide consistency and reduce bias among commercial poultry feed samples. PEG and 1-Day Direct extraction methods are also sufficient but will require standardization in the future.

Technical Abstract: There is no particular DNA extraction kit available for animal feed due to the wide range of variability in feed composition. An optimized DNA extraction technique associated with feed is necessary for comparable downstream sequencing analyses. In this two-part study, the variation in microbiota of five DNA extraction techniques (Part I ) and the microbial compositional diversities of nine commercial poultry feeds (Part II ) were compared. DNA extraction methods included Qiagen QIAamp DNA Stool Mini Kit (Qiagen), modified Qiagen with Lysing Matrix B (MQ), modified Qiagen with celite purification (MQC), polyethylene glycol (PEG), and 1-Day Direct. Genomic DNA was amplified using custom dual-indexed primers and sequenced on an Illumina MiSeq. Sequencing data were analyzed in QIIME2-2021.4 with alpha and beta diversity being determined using Kruskal-Wallis and ANOSIM. Compositional taxonomic differences were determined using analysis of composition of microbiomes (ANCOM). Main and pairwise effects were considered significant at P = 0.05 and Q = 0.05. Results of Part I suggest that there is a direct impact on the diversity and compositional differences based on the extraction method used. Extraction of feed with the Qiagen method produced the least evenness and richness compared to the other protocols used, MQ, MQC, PEG, and 1-Day Direct (P < 0.05). Bead beating and lysozyme application were used in the 1-Day Direct and PEG protocols which produced greater bacterial diversities (P < 0.05). PEG and 1-Day Direct produced numerically greater relative abundance of different genera and phyla associated with extraction methods. Although the use of the Qiagen kit did not result in an increased compositional diversity, application of these kits with adjustments, such as MQ and MQC, can provide consistency and reduce bias among commercial poultry feed samples. PEG and 1-Day Direct extraction methods are also sufficient but will require standardization in the future.