Computational protocol: Effect of Saccharomyces boulardii and Mode of Delivery on the Early Development of the Gut Microbial Community in Preterm Infants

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Protocol publication

[…] Unmapped BAM files were converted to FastQ files using Picard’s SamToFastq []. Sequences were filtered according to their quality using the FastQ quality filter in the FASTX-Toolkit [], resulting in more than 80% of the filtered sequences’ bases having a quality of 20 or higher. Further steps in the analysis were performed using the software Mothur []. FastQ files were converted to FASTA format. Only sequences with lengths between 200 and 300 bases were kept for further analysis. Chimeric sequences were identified using Uchime [], which was run with standard parameters and our sequence collection as the database. Sequences marked as chimeric were then removed. The remaining 16S rRNA sequences were classified using the Wang method and the SILVA bacterial 16S rRNA database as a template (release 102, []), at a bootstrap cutoff of 80%. The taxonomic profile was created using a modified script from STAMP []. [...] Data visualization, including the percentage of bacterial taxa in each sample, statistical analyses, and principal component analysis (PCA) were performed using R and the graphics package ggplot2 []. The phylogenetic tree was prepared in MEGAN5 []. The circular visualization of OTUs present at each time-point was prepared in GraPhlAn []. Differences between groups in relative bacteria DNA amounts were evaluated by the Mann-Whitney U-test. For statistical analyses of taxonomy, relative abundances of each OTU were computed: the number of sequences assigned to a given OTU was divided by the total number of good quality sequences in the sample. Differences in first and second principal component between two groups (S. boulardii versus placebo and Cesarean section versus vaginal delivery) were determined with Student’s t-test. Differences between time points were determined using repeated measures ANOVA and Student’s pairwise t-test as a post-hoc test. Taxonomic contrasts between two groups (S. boulardii versus placebo and Cesarean section versus vaginal delivery) were determined by the Mann-Whitney U-test. Differences between time points were determined with the Mann-Whitney paired U-test. Gut microbiota with nearly constant relative abundance at operational taxonomic unit (OTU) levels (IQR (abundance) <0.5) were removed from taxonomic analyses. P-values were corrected for multiple hypothesis testing using the Benjamini–Hochberg procedure to control the false discovery rate (FDR) []. Species community α-diversity expressed by Simpson’s Diversity index was calculated in Mothur. Contrasts between two groups (S. boulardii versus placebo and Cesarean section versus vaginal delivery) were determined by Mann-Whitney U-test. Differences between time points were determined using repeated measures ANOVA and pairwise Student’s t-test as post-hoc test. Additionally, a linear model was performed at genus level, with log 10-transformed relative abundances as the response variable and S.boulardii intake, mode of delivery, and time as predictors. P-values associated with predictors were also corrected for multiple hypothesis testing using the Benjamini–Hochberg procedure to control the FDR. The power analysis was conducted in GPower 3.1 []. The total bias was calculated as described by Brooks et al. []. Briefly, if A was the mixing ratio of a given bacterium in a mock community dataset and B was the observed proportion, the bias was the difference A−B. A negative value denoted that the bacterial signal was suppressed, while a positive value denoted that the signal was amplified. […]

Pipeline specifications

Software tools Picard, FASTX-Toolkit, mothur, UCHIME, STAMP, Ggplot2, MEGAN, GraPhlAn
Applications Miscellaneous, Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Saccharomyces cerevisiae, Homo sapiens, Bacteria, Firmicutes, Bacteroidetes