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.