Fig 1.
Fecal DNA extraction efficiency varies dependent on extraction method and host species.
Mean total amount (± standard deviation) of DNA extracted from zebrafish gastrointestinal tract (A) or a standardized mass of feces from mice (B), cats (C), dogs (D), or horses (E), using four commercially available DNA extraction kits and one manual extraction procedure (isopropanol). n = 40 individual zebra fish, and 8 individuals for mouse, cat, dog, and horse with 8 samples used for all extraction methods. Samples were extracted and total DNA was measured by fluorometry. Statistical significance determined using one way ANOVA with Student Newman-Keuls post hoc test. Significance defined by p≤0.05 and denoted by like lower case letters, i.e., samples marked with the same letter are significantly different. Whiskers denoting lower standard deviation are cropped at zero.
Table 1.
Comparison of DNA extraction methods.
Cost of DNA extraction methods calculated on per sample basis to include cost of kits, Eppendorf tubes, stainless steel beads, and pipette tips. Time of extraction method determined from start of fecal processing to DNA elution. Mean 260/280 and 260/230 nm absorbance (as determined by spectrophotometry) and standard deviation for all DNA extraction methods for each animal species. Number of amplified samples determined based on the total number of samples resulting in greater than 10,000 reads. n = 8 per extraction method.
Fig 2.
Receiver operator curves of 260/280 and 260/230 nm absorbance for all DNA extraction methods for each animal species.
Absorbance values of all DNA samples from each species (n = 40) were plotted in an ROC generated by Sigma-Plot.
Fig 3.
Comparison of DNA extraction method on next generation sequencing (NGS) relative abundance at the phylum level.
Gray bars represent samples that resulted in sequencing below 10,000 reads. n = 8 samples per extraction method.
Fig 4.
Comparison of DNA extraction method on next generation sequencing (NGS) relative abundance at the family level.
Gray bars represent samples that resulted in sequencing below 10,000 reads. n = 8 samples per extraction method.
Fig 5.
Principal Component Analysis (PCA) of samples with successful amplification and sequencing in at least half (4/8) samples.
Colors denote extraction method: DNeasy (blue), PowerFecal (pink), CadorPathogen (purple), QIAmp Stool (green), and Isopropanol (brown). Numbers denote individual animal samples tracked across all kits tested for that species
Fig 6.
Diversity of fecal microbiota.
Chao1 estimate of microbial diversity plotted by Tukey box and whisker graph. For zebrafish and horse samples, statistical significance was determined using student’s t-test. For mouse, cat, and dog samples, statistical significance was determined using ANOVA with Student Newman Keuls post hoc test. Statistical significance defined by p≤0.05 and denoted in the figure by lower case letters.