horse_5_rep1_camelina
The V3V4 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene was targeted for amplification via PCR. Amplicons were obtained by using the universal Illumina primer couple (https://support.illumina.com/documents/documentation/chemistry documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). Each 25 L PCR reaction contained 12.5 L of 2 Kapa HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA, USA), 5 L of each primer (at 1 M concentration), and 2.5 L of template DNA. Thermal cycling conditions consisted of an initial denaturation, followed by 25 cycles at 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s, with a final extension step at 72 C for 5 min.  After amplification, the PCR amplicons were purified using Agencourt AMPure beads (Beckman Coulter, Milan, Italy) to remove any residual primers and impurities. The purified PCR products were then indexed (barcoded) using the Nextera XT Index Kit Set A (Illumina Inc., San Diego, CA, USA) in accordance with the manufacturer instructions. The success of the amplification and indexing was verified on an Agilent TapeStation 4200 using a D1000 ScreenTape assay, which confirmed the expected amplicon size (approximately 630 bp) and the absence of primer-dimer artifacts.  Following quality control and quantification, the indexed libraries were singularly normalized to 4 ng/L. Equal volumes of these normalized libraries were pooled to achieve the required molarity for sequencing, and a PhiX Control v3 library (Illumina) was used as spike-in for the pool at 30% of the total volume, as an internal sequencing control to increase diversity. The final pooled library, along with the appropriate buffers and sequencing reagents, was loaded onto an Illumina MiSeq2 platform. Sequencing was performed using a MiSeq Reagent Kit v3 in a 2  300 bp paired-end run.