16S Seq of CF Sputum
DNA libraries were prepared by the  University of Michigan Microbial Systems Molecular Biology Laboratory. In brief, the V4 region of the bacterial  16S rRNA gene was amplified using touchdown PCR with barcoded dual-index  primers. The touchdown PCR cycles  consisted of 2 min at 95C, followed by 20 cycles of 95C for 20 sec, 60C  (starting from 60C, the annealing temperature decreased 0.3C each cycle)  for 15 sec and 72C for 5 min, followed by 20 cycles of 95C for 20 sec, 55C  for 15 sec and 72C for 5 min and a final 72C for 10 min. Resulting amplicon libraries were  normalized and sequenced on an Illumina sequencing platform using a MiSeq  Reagent Kit V2 (Illumina, San Diego, CA, USA). The final load concentration was 4.0-5.5 pM  with a 15% PhiX spike to add diversity.