Computational protocol: Serotonin Transporter Genotype Modulates the Gut Microbiota Composition in Young Rats, an Effect Augmented by Early Life Stress

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Protocol publication

[…] DNA was extracted from fecal samples using the PowerFecal DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, United States) following the manufacturer’s protocol. DNA concentration and purity (260/280 and 260/230 ratios) was quantified using NanoDrop 2000c (Thermo ScientificTM) and samples were thereafter stored at -20°C until further use.In triplet a total of 10 ng μL-1 of extracted DNA per sample was used for PCR-amplification of a 400 bp fragment of the bacterial 16S rRNA gene using a primer set with a sample specific barcode sequence (see Table for details). The following 25 μL master mix was used: 200 nM dNTPs mix, 1X HF-Buffer, 500 nM forward and reverse primers, 0.5 U Phusion DNA polymerase (ThermoscientificTM). To ensure the specificity of the reaction, the following PCR conditions were set: initial denaturation step at 98°C for 30 s, followed by 30 cycles of 98°C for 10 s, 70°C for 30 s, 72°C for 30 s; with a final step of 72°C for 7 min. After amplification the triplet PCR mixtures were pooled and loaded on a 2% agarose gel stained with SYBR-safe (ThermoscientificTM) and the 400 bp fragments were excised. DNAs were extracted from the excised agarose parts using the Qiaex II gel extraction kit (Qiagen, the Netherlands) following the manufacturer’s protocol. Purified fragments were eluted in 27 μL of Ultrapure water. Final concentration and purity was determined using the nanodrop 2000c and dsDNA concentration was quantified using Quan-iT Picogreen dsDNA reagent (Life Technologies, the Netherlands).Prior to sequencing, a total of 96 samples were pooled at equimolar concentrations resulting in a total of 2 μg dsDNA of 40 ng μL-1. The amplicon pool was send to Genewiz, United Kingdom and sequencing was carried out on an Illumina MiSeq instrument 2 bp × 300 bp. Illumina Mi-Seq raw data were paired, demultiplexed and processed using the Quantitative Insights Into Microbial Ecology toolkit (). In brief, 16S rRNA bacterial partial sequences were quality trimmed using the default parameters in QIIME and reads were then binned into operational taxonomic units (OTUs) at 97% sequence identity using open-reference OTU picking method in QIIME. A representative sequence for each phylotype was aligned against the Greengenes corset () using PyNAST (), with sequences classified using the Greengenes taxonomy via blast. The alignment was filtered to remove common gaps and a phylogenetic tree was constructed using FastTree (). For all OTU-based analyses, the original OTU table was rarified to a depth of 6000 sequences per sample (the fewest in a single sample) to minimize effects of sampling effort on the analysis. The Quantitative Insights Into Microbial Ecology toolkit was also used to generate weighted/unweighted UniFrac distance matrices () and alfa-diversity metrics, including OTU richness (unique OTUs), ChaoI richness estimation, and Faith’s phylogenetic diversity indices. All data are presented as mean ± SEM. The microbiome composition was analyzed using the Wilcoxon rank test using the statistical software package SPSS 21.0 (IBM). The rank test-Kruskal–Wallis test was used for comparison of abundant taxa. The statistical significance was indicated as follows: ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001.Random forest identifies the subset of most relevant features by constructing a collection of decision trees. Constructing trees incorporating only a random subset of the features, which in turn avoids overfitting, control variance. The random forest package for R (v4.6-7) was used with default settings and baseline error was calculated as previously described (). […]

Pipeline specifications

Software tools QIIME, PyNAST, FastTree, UniFrac
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Diseases Brain Diseases, Gastrointestinal Diseases, Intestinal Diseases
Chemicals Serotonin