bird 8 intestine triplicate 3
The V3 and V4 regions of the 16S rRNA gene were amplified through PCR from the extracted DNA using the primers 341F: ACTCCTACGGGAGGCAGCAG [22] and 806R: GGACTACHVGGGTWTCTAAT [23]. Furthermore, two PCR setups were used as some samples did not amplify, and others were overamplified. The following PCR setup was used for the garum samples: 95C for 10 min, followed by 32 cycles of 95C for 15 sec, 56C for 20 sec, and 72C for 40 sec. This was followed by a single cycle of 72C for 10 min, whereafter it was stored at -20C until further processed. Samples from the crop, stomach, and intestines had the following PCR setup: 95C for 10 min, followed by 32 cycles of 95C for 15 sec, 56C for 20 sec, and 72C for 40 sec. Followed by a single cycle of 72C for 10 min and then stored at -20C until further processed. During this process, we added six PCR controls, leaving us with a total of 124 samples for the downstream library build. PCR products were resolved with gel electrophoresis and stored at -20C. The PCR product was pooled to make amplicon libraries with Illumina TrueSeq DNA PCR-free library, with each PCR product equally represented by assessing the gel electrophoresis results and dividing the samples into groups depending on the results. The library was built on Ilumina TrueSeq DNA PCR-free Library following the manufacturers instructions, and the final libraries were sent for Illumina MiSeq v3 2 x 300 bp paired-end sequencing. Negative control, DNA extraction kit controls, and PCR product controls were included throughout all stages of the laboratory process. The samples were sequenced a second time to ensure enough data for further processing.