title: metabarcoding of cow diary milk
design: Samples were thawed at 4C for 16 hours and total DNA was extracted from 20 ml in phenol/chloroform according to Ponzoni et al. (2009). As a minor modification of the original method, the first incubation step was conducted at 65C for 6 hours under constant agitation.  Plant DNA was extracted from technical controls using the DNeasy Plant mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Purity and quantity were determined with Nanodrop Spectrophotometer 2000 (Thermofisher Scientific, Wilmington, DE, USA). RuBisCO gene amplicon molecular identification. To confirm the presence of plant nucleic acid in the total DNA extracted from milk, a 351bp RuBisCO gene fragment was amplified with the following primers: RUB F2 TTGGCAGCATTCCGAGTAAC and RUB R2 GTGAGGCGGACCTTGGAAAG by using the GeneAmp PCR System 9700 (Applied Biosystems FosterCity, CA). The PCR condition included a denaturation step at 95 C for 15, followed by 35 cycles of 94 C for 30, 57 C for 90 and 72 C for 90 followed by a final elongation at 72 C for 10. Fragments were visualised on the Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and only samples with positive amplification were submitted to DNA metabarcoding analysis. Library preparation and sequencing DNA samples were amplified in two steps, as follows: an initial PCR amplification of the variable rbcL region of the RuBisCO gene was conducted using locus specific primers with the following Illumina adapter overhangs: rbcL F 5- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGTCACCACAAACAGAGACTAAAGC-3 and rbcL R 5- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTAAAATCAAGTCCACCRCG -3 and a subsequent amplification that integrates relevant flow-cell binding domains and unique indices (NexteraXT Index Kit, FC1311001/FC1311002). Libraries were sequenced on MiSeq instrument (Illumina, San Diego, CA) using the 300 bp paired-end mode.