processed removing the sequence reads of too low quality (only "passing filter" reads were selected) and discarding reads containing adaptor sequences or failing PhiX Control with an in-house filtering protocol. A quality assessment on the remaining reads was performed using the FASTQC quality control tool version 0.10.0. Illumina sequencing data analysis Multiplexed FASTQ files as provided by BaseClear were first used to generate Illumina paired-end sequence pseudoreads by PEAR [E5], using the default settings. For gene sequencing analysis, a customized Python workflow based on Quantitative Insights Into Microbial Ecology (QIIME version 1.8) [E6] was adopted (http://qiime.org). Reads were filtered for chimeric sequences using the UCHIME algorithm version 4 [E7]. Hierarchical clustering of samples was performed using UPGMA with weighted UniFrac as a distance measure as implemented in QIIME 1.8. Figures resulting from these clustering analyses were generated using the interactive tree of life (iTOL) tool [E8]. The Ribosomal Database Project classifier version 2.3 was performed for taxonomic classification of the sequence reads [E9]. Alpha diversity metrics (PD whole tree, Chao1, Observed Species and Shannon) were calculated by bootstrapping 4822 reads per sample, and taking the average over 10 trials. Chao1 is a species richness metric; whereas PD whole tree is a species diversity metric (defined as a combination of both species richness and species evenness) comparable to Shannon but additionally taking taxonomical distance into account. For visualization of the differential microbiome, Cytoscape software version 3.1.3 [E10] was used together with developed Python scripts for generating the appropriate input data deriving from the QIIME analysis.
processed removing the sequence reads of too low quality (only "passing filter" reads were selected) and discarding reads containing adaptor sequences or failing PhiX Control with an in-house filtering protocol. A quality assessment on the remaining reads was performed using the FASTQC quality control tool version 0.10.0. Illumina sequencing data analysis Multiplexed FASTQ files as provided by BaseClear were first used to generate Illumina paired-end sequence pseudoreads by PEAR [E5], using the default settings. For gene sequencing analysis, a customized Python workflow based on Quantitative Insights Into Microbial Ecology (QIIME version 1.8) [E6] was adopted (http://qiime.org). Reads were filtered for chimeric sequences using the UCHIME algorithm version 4 [E7]. Hierarchical clustering of samples was performed using UPGMA with weighted UniFrac as a distance measure as implemented in QIIME 1.8. Figures resulting from these clustering analyses were generated using the interactive tree of life (iTOL) tool [E8]. The Ribosomal Database Project classifier version 2.3 was performed for taxonomic classification of the sequence reads [E9]. Alpha diversity metrics (PD whole tree, Chao1, Observed Species and Shannon) were calculated by bootstrapping 4822 reads per sample, and taking the average over 10 trials. Chao1 is a species richness metric; whereas PD whole tree is a species diversity metric (defined as a combination of both species richness and species evenness) comparable to Shannon but additionally taking taxonomical distance into account. For visualization of the differential microbiome, Cytoscape software version 3.1.3 [E10] was used together with developed Python scripts for generating the appropriate input data deriving from the QIIME analysis.