ITS sequencing of human sample
We utilized the Powersoil (MoBio) extraction kit to isolate microbial DNA. ETA samples are highly viscous and required pretreatment with Dithiothreitol (0.1% DTT in phosphate-buffered saline) in 1:1 dilution in order to dissolve the mucus to allow for the use of DNA extraction columns. We then amplified the microbial DNA by PCR using the method of Caporaso et al. and the Q5 HS High-Fidelity polymerase (NEB) regions 1 and 2 of the internal transcribed spacer (ITS) rRNA gene. Four microliters per reaction of each sample were amplified with a single barcode in triplicate 25 microliter reactions. In order to obtain an adequate number of amplicons, we employed a 2-step nested PCR protocol. The initial cycle conditions were 98C for 30 seconds, 15 cycles of 98C for 10 seconds, 55C for 30 seconds and 72C for 90 seconds followed by 72C for 2 minutes. We utilized 5 microliters of the initial PCR reaction in the second cycle and were processed using the following conditions: 98C for 30 seconds, 15 cycles of 98C for 10 seconds, 55C for 30 seconds and 72C for 1 minute followed by 72C for 2 minutes. Amplified DNA was then purified using AMPure XP beads (Beckman) at a 0.7:1 ratio (beads:DNA). A pooled sample was created by combining 30 microliters of each sample and was further purified using the MinElute PCR purification kit. A second purification of the sample using AMPure XP beads and a final cleanup using the Purelink PCR Purification Kit (Life Technologies) were performed. The purified pool was quantitated in triplicate through high-resolution microfluidics-based automated electrophoresis using the 2100 Bioanalyzer instrument (Aligent). The sequencing pool was prepared according to instructions by Illumina with an additional incubation at 95C for 2 minutes immediately following the initial dilution to 20 picomolar.33 The sequencing pool was then diluted to a final concentration of 7 pM + 15% PhiX control. We utilized the Illumina Miseq platform for sequencing of the amplicons.  Reagent controls were included for DNA extraction and PCR amplification.  Sequencing reads were demultiplexed into individual sample/replicate fastq files.