16S RNA seq of human breastmilk in women with and without T1D
The V4 region of the 16S rRNA gene was amplified using primers 515F/806R for bacterial analysis. Each sample was amplified in duplicate by PCR and the PCR products were visualised by agarose gel electrophoresis and quantified using Agilent 4200 TapeStation System. Equimolar amounts of purified amplicons were pooled and both PCR replicates were independently sequenced to ensure sufficient DNA yield using the Illumina MiSeq platform with version 3 600 cycle (2300 bp) kit. Samples were sequenced in three different sequencing runs (runs 11, 16 and 22).