## 3 Results
### Optimized Extraction Use for Fecal Microbiome  
The fecal microbiome is an increasingly important focus in domestic animal health and human medicine [39; 40; 41; 42; 43]. We compared our NaOH extraction method to a commercial kit for extraction of DNA suitable for PCR amplification of the V4 region of 16S rDNA for microbiome analyses from swine fecal swab samples. The analysis was for six samples including two different growth ages using duplicate swabs for the two extraction methods. Paired swabs were extracted using either a commercial kit or the NaOH extraction with paramagnetic bead capture. Quantification of the PCR products from the NaOH extraction and NanoDrop results from kit extraction are provided in Table 4. The NaOH extraction with magnetic bead capture was comparable, if not superior, to the commercial kit based on the quantification of PCR products (Table 4). The pipeline for analysis of the sequence data (see Methods) has been described [28]. Figure 5 shows the alpha and beta diversity for the swab microbiome. Kruskal-Wallis test results show no significant differences between alpha diversities for the two extraction methods (see \(p\) values in Figure 5). Analysis of similarity (ANOSIM) conducted in qime2 also shows that microbiome communities of these two methods were very similar. The swine gut microbiome structure based on Bray-Curtis distance (Figure 5C) and membership as measured by Jaccard distance (Figure 5D) were separated by animal growth stage for lactation versus nursery (ANOSIM, R = 0.962, \(p\) = 0.003 for Bray-Curtis distance; R = 0.998, \(p\) = 0.001 for Jaccard distance) but not by DNA extraction methods (ANOSIM, R = \(-\)0.081, \(p\) = \(-\)0.767 for Bray-Curtis distance; R = \(-\)0.120, \(p\) = 0.832 for Jaccard distance). Figure 6 shows the comparisons of the microbiome composition at the phylum and genus levels for the pairs of swabs from the two ages. Although there are differences between individual pigs for the minor phyla, especially at 10 days of age, the paired samples show similar distributions and frequencies whether extracted with the kit or with the NaOH with paramagnetic bead capture. There are more differences between the extraction methods for the genus-level analyses, but the data clearly support the NaOH extraction with magnetic bead capture as an inexpensive alternative for high-throughput microbiome analyses.  
Figure 4: Average Ct values from qPCR-HRM for NaOH extracts from three samples from sewage water (A–C) or pond water (D–F). Aliquots from each extract were subjected to paramagnetic bead purification (open bars) or dilution with 4 volumes of Te (gray bars). Final samples for each method were adjusted to the same volume and assayed by qPCR-HRM in triplicate. Error bars are standard deviation. Ct values for controls: 0.25 ng 908 DNA—16.1 \(\pm\) 2.5; H\({}_{2}\)O—32.4 \(\pm\) 0.45 (no melt product for 16S product).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**ID**} & \multirow{2}{*}{**Pig Age (Day)**} & \multicolumn{2}{c}{**PCR Product (ng/uL)**} \\ \cline{3-4}  & & **Kit** & **NaOH + MB** \\ \hline
1 & 10 & 241 & 27 \\
2 & 10 & 68 & 5739 \\
3 & 10 & 22 & 410 \\
4 & 59 & 55 & 52 \\
5 & 59 & 82 & 216 \\
6 & 59 & 94 & 150 \\ \hline \hline \end{tabular}
\end{table}
Table 4: DNA quantification from duplicate fecal swabs from each of six pigs of two growth stages, day 10 lactation or day 59 end of nursery. Pairs of swabs were extracted either with a commercial kit (Kit) or the NaOH extraction with magnetic bead capture (NaOH + MB). NaOH extraction was quantified by qPCR; kit extraction was quantified with a NanoDrop.  
Figure 5: Microbiome alpha diversity (**A**,**B**) and beta diversity (**C**,**D**) from six pig fecal samples extracted either with a kit (circle) or the NaOH (triangle) method. Samples numbers are the same as in Table 4, and the paired samples are represented with the same color and connected with a line.