CF Sputum Microbiome
Amplification conditions were: 2 minutes at 94 C; 30 cycles of 30 seconds at 94 C, 30 seconds at 56 C or 58 C, and 30 seconds at 72 C; followed by 5 minutes final extension at 72 C. Libraries were loaded onto the Illumina cBot cluster station for cluster generation. First strand sequencing began with the Illumina sequencing primer for eight cycles to read the barcode, followed by a denaturation chemistry and hybridization with a mix of the V5 forward and V7 reverse primers at 5  M final concentration each. After 36 cycles of sequencing, another round of denaturation was performed and a mixture of V6 forward and V6 reverse primers (5  M each) was hybridized for another 36 cycles of sequencing.