16S rRNA sequencing of bovine: semen
Samples were sent to Zymo Research Corporation (Irvine, CA) for 16S rRNA microbial analysis. DNA extraction for preputial swabs and semen samples were conducted using 250 L of sample and the ZymoBIOMICS DNA Miniprep Kit or the ZymoBIOMICS-96 MagBead DNA Kit (Zymo Research, Irvine, CA), respectively. The V3-V4 region of the bacterial 16S rRNA gene was amplified, and the samples were barcoded using the library Quick-16S NGS Library Prep Kit (Zymo Research, Irvine, CA). The barcoded amplicons were quantified with real-time polymerase chain reaction (qPCR) fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator (Zymo Research, Irvine, CA), then quantified with TapeStation(Agilent Technologies, Santa Clara, CA) and Qubit (Thermo Fisher Scientific, Waltham, WA). The ZymoBIOMICS Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction. The ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process. The final library was sequenced on Illumina Nextseq (Illumina, San Diego, CA, USA) with a P1 reagent kit (600 cycles). The sequencing was performed with 30% PhiX spike-in.