title: Evaluating DNA Extraction Methods for Community Profiling of Pig Hindgut Microbial Community.
abstract: Recovery of high quality PCR-amplifiable DNA has been the general minimal requirement for DNA extraction methods for bulk molecular analysis. However, modern high through-put community profiling technologies are more sensitive to representativeness and reproducibility of DNA extraction method. Here, we assess the impact of three DNA extraction methods (with different levels of extraction harshness) for assessing hindgut microbiomes from pigs fed with different diets (with different physical properties). DNA extraction from each sample was performed in three technical replicates for each extraction method and sequenced by 16S rRNA amplicon sequencing. Host was the primary driver of molecular sequencing outcomes, particularly on samples analysed by wheat based diets, but higher variability, with one failed extraction occurred on samples from a barley fed pig. Based on these results, an effective method will enable reproducible and quality outcomes on a range of samples, whereas an ineffective method will fail to generate extract, but host (rather than extraction method) remains the primary factor.
authors.affiliation: Advanced Water Management Centre, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia.
authors.forename: Yang
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences and Institute of Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia.
authors.forename: Philip
authors.initials: P
authors.lastname: Hugenholtz
authors.affiliation: Advanced Water Management Centre, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia.
authors.forename: Damien John
authors.initials: DJ
authors.lastname: Batstone
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D020869:Gene Expression Profiling; D023281:Genomics / Q000379:methods; D008828:Microbiological Techniques; D064307:Microbiota; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013552:Swine / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S