16S amplicon sequencing of bacteria in dairy cow's milk
Milk samples collected from all 4 mammary gland quarters at calving and on days 1, 3, 5, 7, 10, 14, 17, 21 and 28 of lactation. A sampling control (Parlour Control) taken on all sampling occasions. DNA was extracted from 1599 milk samples using a Qiagen QIAamp 96 DNA Blood kit with additional enzymatic steps (lysozyme, lysostaphin and mutanolysin). An extraction control was processed with every DNA extraction run. PCR with general bacterial primers 27F and 534R with overhanging sequence to facilitate MiSeq sample preparation, including PCR and model community controls (Staphylococcus aureus, E. coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis) in every PCR run. All samples and controls sequenced in eight 300 bp paired end sequence runs.