Microbiota composition was determined of sow feces, sow vaginal swabs and piglet jejunal digesta. Samples were frozen on dry-ice and stored at -80°C. To isolate DNA, samples were mixed in a 1:1 ratio with phosphate buffered saline (PBS) and centrifuged for 5 min at 4°C at 300xg. Supernatant was collected and centrifuged for 10 min at 4°C at 9000xg. DNA was extracted from the pellet using the “QIAamp DNA stool minikit” (Qiagen, Valencia, CA, USA) according to manufacturers’ instructions, after mechanical shearing of the bacteria in Lysing Matrix B tubes using the FastPrep-24 (MP Biomedicals, Solon, OH, USA). Quality and quantity of DNA were checked using the NANOdrop (Agilent Technologies, Santa Clara, CA, USA). PCR was used to amplify (20 cycles) the 16S rRNA gene V3 fragment using forward primer V3_F (CCTACGGGAGGCAGCAG) and reverse primer V3_R (ATTACCGCGGCTGCTGG). PCR efficiency was checked on agarose gel. Amplicons were sequenced using paired-end, excluding one sample that did not pass the quality control, 150bp technology on a MiSeq sequencer (Illumina, San Diego, CA, USA) at a sequencing depth in the range of 196K-1.2M read-pairs per sample (median 670,647 read-pairs per sample).
|Date made available||2 Mar 2020|