16S V4 sequencing of cf lung microbiome
The V4 region of the resulting DNA was amplified with the 16S universal primers 515F (5-GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3). A single-step 30 cycle PCR integrating sequencing amplification and library adapter/barcode attachment was performed using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) by first incubation at 94 C for 3 minutes, followed by 28 cycles of 94 C for 30 seconds, 53 C for 40 seconds and 72 C for 1 minute, followed by a final elongation step at 72 C for 5 minutes. Amplification products were then normalized, pooled and purified using calibrated Ampure XP beads for Illumina Miseq sequencing.