Computational protocol: The effects of inhaled aztreonam on the cystic fibrosis lung microbiome

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Protocol publication

[…] Microbial community profiling of sputum samples was conducted as previously described []. Amplification of the V3 hypervariable region of the 16S ribosomal RNA (rRNA) gene was carried out using reverse and forward barcoded primers using the Illumina MiSeq technology at the McMaster Genome Facility (Hamilton, ON). Reagent blanks are run for each set of DNA extractions, and samples are excluded if these controls are positive for PCR products and DNA extractions repeated as necessary. Sequencing reads were analyzed using custom Perl scripts []. Briefly, sequences with low quality reads were trimmed using Cutadapt []. PANDAseq was used to assemble paired-end reads []. Operational taxonomic units (OTUs) were picked using AbundantOTU+ for OTUs with ≥97% identity []. The RDP classifier (version 2.2) was used to assign taxonomy against the Greengenes reference database [, ]. Analysis was carried out using QIIME (version 1.8.0) and Phyloseq (version 1.16.2) R package [, ]. [...] Microbial communities were assessed based on the below discrete categorical variables. Samples collected prior to treatment (pre) were compared with those collected after treatment (post) to determine how AZLI impacts the microbiome of a naïve patient population. Post-samples were further stratified based on whether they were collected during “on” vs “off” cycled AZLI therapy. Those samples collected within 7 days of parenteral antibiotics for a pulmonary exacerbation were assessed for changes associated with systemic therapies and excluded from other analyses to limit confounding effects []. In order to determine if there are biomarkers associated with clinical response to AZLI, the microbiome of responders vs non-responders was also compared. Finally, we analyzed whether there were gender-based differences in the microbiome as an attempt to better characterize the gender gap in CF lung disease []. Wilcoxon rank-sum tests and paired t tests were used to test for significant differences in alpha diversity measures of observed OTUs and Shannon diversity indices (SDI), in the phyloseq package in R [, ]. Community structures were assessed using Bray-Curtis (BC) beta diversity measures after proportionally normalizing all samples as previously described []. Permutational multivariate analysis of variance (PERMANOVA) was used to analyze statistical differences in beta diversity using the vegan package in R []. Results were visualized using principal coordinate analysis (PCoA) plots. In addition to community-wide differences, we were also interested in differences at the genus and OTU level; Wilcoxon rank-sum tests and paired t test were employed to calculate differences in the relative abundance values of organisms present in >20% of samples for the different categorical variables being assessed. Log abundance plots were used to visualize these results using ggplot2 package in R []. Samples with an abundance value of 0 were changed to a value of 1 since log(0) is undefined and log(1) = 0. As a sensitivity analysis, a linear mixed model with random intercepts was constructed to examine differences in diversity as well as relative abundance of organisms present in >20% of samples (similar to above) for the pre- and post-AZLI period. Secondly, a restricted analysis (using only one sample/patient/category) of the observations closest to initiation of AZLI in the pre- and post-periods was conducted utilizing paired t tests and Wilcoxon rank-sum tests for the previously detailed tests as a comparator. […]

Pipeline specifications

Software tools cutadapt, PANDAseq, RDP Classifier, QIIME, phyloseq, vegan, Ggplot2
Databases Greengenes
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Homo sapiens, Pseudomonas aeruginosa
Diseases Cystic Fibrosis, Infection, Pseudomonas Infections
Chemicals Aztreonam