Computational protocol: Fast acquisition of a polysaccharide fermenting gut microbiome by juvenile green turtles Chelonia mydas after settlement in coastal habitats

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Protocol publication

[…] DNA was extracted from a subsample of 0.25 g from each fecal or rectal sample using the PowerSoil DNA kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer’s instructions. All DNA extracts were kept frozen at − 20 °C until further analysis. Massive bar-coded 16S rRNA gene-based libraries in the Eubacteria domain were sequenced by using the MiSeq Illumina platform (Molecular Research DNA LP, Shallowater, USA). These gene libraries were constructed by targeting the V1–V3 hypervariable regions with the primer set 27F (5′-AGRGTTTGATCMTGGCTCAG-3′)/519R (5′-GTNTTACNGCGGCKGCTG-3′) as previously described in []. The obtained DNA reads were compiled in FASTq files for further bioinformatic processing. Trimming of the 16S rRNA barcoded sequences into libraries was carried out using QIIME software version 1.8.0 []. Quality filtering of the reads was performed at Q25, the default set in QIIME, prior to the grouping into operational taxonomic units (OTU) at a 97% sequence homology cutoff. The following steps were performed using QIIME: Denoising of sequence data using Denoiser [], picking up of OTU reference sequences via the first method of the UCLUST algorithm [] and, for sequence alignment and chimera detection, processing by PyNAST [] and ChimeraSlayer []. OTUs were then taxonomically classified using BLASTn against GreenGenes and RDP (Bayesian Classifier) databases and compiled into each taxonomic level []. [...] A general lineal model (GLM) using locality (Ubatuba vs. Praia do Forte) as a fixed factor and turtle curved carapace length as a covariable was used to test the hypothesis that the microbial diversity of wild green turtles increases with turtle size and varies across localities. A general lineal model using origin (captive vs. wild) as a fixed factor and turtles curved carapace length as a covariable was used to test the hypothesis that the microbial diversity of green turtles increases with turtle size and differs between captive and wild green turtles from subtropical Ubatuba. GLMs were run in IBM SPSS Statistics 23. Multivariate principal coordinate analysis (PCoA) based on Bray-Curtis similarity distances was carried out on the OTUs incidence matrix using the CANOCO software package, version 5 (Microcomputer Power, Ithaca, NY, USA), to identify clusters of green turtles differing in the community structure of their microbiomes. […]

Pipeline specifications

Software tools QIIME, UCLUST, PyNAST, ChimeraSlayer, BLASTN, SPSS
Databases Greengenes
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Chelonia mydas, Bacteria, Bacteroidetes, Firmicutes, Lachnospiraceae, Danio rerio