title: DIETARY HABITS AND RELATIONSHIP WITH THE PRESENCE OF MAIN AND TRACE ELEMENTS, BISPHENOL A, TETRABROMOBISPHENOL A, AND WITH THE LIPIDIC, MICROBIOLOGICAL AND IMMUNOLOGICAL PROFILES OF BREAST MILK
design: The V3-V4 hypervariable region of the 16S rRNA was amplified by PCR and sequenced as previously described (Klindworth et al., 2013). Briefly, universal primers S-D-Bact-0341-b-S-17 (CCTACGGGNGGCWGCAG) and S-D-Bact-129 0785-a-A-21 (GACTACHVGGGTATCTAATCC) were used to amplify the V3-V4 hypervariable region of the 16S rRNA and then, barcodes were appended to 3 and 5 terminal ends of the PCR amplicons in a second PCR-reaction in order to allow separating forward and reverse sequences. The pooled, purified and barcoded DNA amplicons were sequenced using the Illumina MiSeq 2 x 300 bp pair-end protocol (Illumina Inc., San Diego, CA, USA) following the manufacturers recommendations at the facilities of Parque Cientfico de Madrid (Tres Cantos, Spain) (Klindworth et al., 2013).