title: 16s-seq from preterm gut
design: Purified DNA samples were qualitatively assessed for integrity by agarose gel electrophoresis, and concentration was estimated using the Nanodrop 8000. The 16S rRNA V3V4 hypervariable region was amplified with the Bakt_341F and Bakt_805R primers 22. Each PCR reaction was performed in a final volume of 50 L, containing 25 L of 1x Platinum Multiplex PCR Master Mix, 100 nM of each forward and reverse primer, and 100 ng of bacterial DNA, using a Thermo ABI GeneAmp 9800 Fast Thermal Cycler. The thermal cycling conditions were optimized as follows: 95C for 4 min, followed by 25 cycles of 95C for 30 s, 55C for 30 s, and 72C for 3 min. The PCR products were verified for integrity by agarose gel electrophoresis, and their concentrations were measured using the Nanodrop 8000. DNA libraries were sequenced at the Sequencing Unit of the Instituto Nacional de Medicina Genmica on a MiSeq Illumina platform  using the MiSeq Reagent v3 Kit.