Computational protocol: Microbial colonization is required for normal neurobehavioral development in zebrafish

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Protocol publication

[…] The MiSeq reads were processed using mothur (v1.391). Reads were filtered by Phred quality (Q30 with 50 nucleotide window length) and removed if they failed to form complete contigs. Read-pair contigs were removed if they contained an ambiguous base call (0 allowed), homopolymers (maximum of 8 nucleotides), were greater than 275 nucleotides, or failed to align to the V4 region of the Silva 16S rRNA gene reference alignment (v128). A preclustering algorithm was used to denoise the read-pair contigs. Chimeras were identified and removed using the UCHIME algorithm of USEARCH software. A Bayesian classifier and the Ribosomal Database Project (RDP, training set version 16) were used to classify the read-pair contigs with a minimum bootstrap of 80%. Read-pair contigs that did not classify at the level of kingdom or that classified as Archaea, Eukaryota, chloroplasts, or mitochondria were removed from further analysis. An operational taxonomic unit (OTU) table with rows and columns representing samples and bacterial taxa counts (binned at 3% dissimilarity) and their taxonomic assignments was generated for subsequent statistical analysis. R-Studio software (v0.99.902) was used to sort the OTU table in descending order by column means and the columns were removed from subsequent analysis if they contained counts in less than 10% of the samples. Alpha and beta diversity analysis was performed using PRIMER 7 software (Primer-E v7.0.11) for total number of species (S), Margalef’s species richness (d), Pielou’s evenness index (J’), Simpson index (i.e., 1-λ’), and Shannon diversity index (i.e., H’), Bray-Curtis similarities, ANOSIM, non-metric multidimensional scaling (NMDS), and heatmap analyses. For Bray-Curtis, ANOSIM, and NMDS, the read-pair contigs were standardized as a percentage per sample. Relative abundance and heatmap analyses was performed on standardized percentages. Heatmap OTU and sample dendrograms were constrained by hierarchical cluster analysis (mode group average) of the Index of Association similarities between OTUs and Bray-Curtis similarities between samples, respectively. Within group and between group Bray Curtis similarities are shown. […]

Pipeline specifications

Software tools mothur, UCHIME, USEARCH
Application 16S rRNA-seq analysis
Organisms Homo sapiens, Danio rerio, Aeromonas veronii, Vibrio cholerae