title: 16S sequencing of human milk
design: 1 ml of milk was centrifuged for 20 minutes at 8000 g, and the pellets were dissolved in 500 ľl of the supernatant. The first set of 30 samples was processed with the DNA Stool Mini kit (QIAGEN, Switzerland) according to the manufacturers instructions, with two modifications: the milk samples were heated at 95°C for 5 s and a bead-beating step with Fastprep (MP Biochemicals, USA) was added (30 s, speed 6 with 0.1 mm silica beads). The second set of 60 samples was processed with the Fast DNA SPIN Kit for soil (MO BIO, USA) according to the manufacturers instructions. For the 16S rRNA gene sequencing, the approach proposed by [Caporaso 2012] was followed with some modifications. The V4 region was amplified by PCR (for 30ul reaction volume: 6ul of DNA, 1.2ul of each primer at 10 pM, 0.6ul of each dNTP at 2.5 mM, 6ul of Expand High Fidelity PLUS Reaction Buffer, 0.3ul of Expand High Fidelity PLUS Enzyme Blend 5U/ul (Roche Applied Science, Switzerland). Amplification conditions were the following: 94°C for 2 min followed by 30 cycles of 94°C for 30 seconds, 50°C for 30 seconds and 72°C for 30 seconds followed by 7 minutes at 72°C and finally held at 4°C). The primers mapped to the conserved regions flanking V4 and produced an amplicon of 425 bp. In addition, they carried a specific barcode to multiplex the samples for sequencing and the Illumina sequencing adapter. The primers were identical to these used in [Caporaso 2012]. After quantification and quality check by capillary electrophoresis (LabChip GX, Perkin Elmer, USA), all the amplicons were pooled in equimolar amount and purified. In cases where the DNA concentration was below the detection limit, all available sample volume was used for pooling. Sequencing was performed on the MiSeq instrument V2.1 (with Hard Coded matrix for data acquisition) paired-end 250 bp (MS-102-2003). Demultiplexing was performed by the MiSeq software (V2.1).