Computational protocol: Host-genotype dependent gut microbiota drives zooplankton tolerance to toxic cyanobacteria

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Protocol publication

[…] This is experiment 2.To determine whether the composition of the gut microbiota differed between tolerant and susceptible genotypes, and whether it was affected by exposure to a cyanobacteria, the gut microbiota of the 24 donor populations described above was characterized through next-generation sequencing of 16S rRNA. These 24 donor populations were the same as those described in experiment 1, which were continuously maintained in the laboratory, under similar conditions as in experiment 1. The gut microbiota characterization of these populations was performed ~1.5 year after the transplant, i.e., after ~58 generations of exposure to cyanobacterial vs. green algal diet.Twenty adult Daphnia were sampled from each donor population and placed in fresh, sterile (i.e., autoclaved) ADaM medium during 24 h, to remove most transient bacteria and food particles from the gut. Their guts were dissected and pooled, and the microbiota was extracted, using the same protocol as described in experiment 1. The 24 samples were stored at −20 °C until further processing.Sequencing library preparation: DNA was extracted using a PowerSoil DNA isolation kit (MO BIO laboratories), and dissolved in 20 µL MilliQ water. The total DNA yield was determined using a Qubit dsDNA HS assay (Invitrogen) on 3 μL of sample. Because of initially low bacterial DNA concentrations in some samples, a nested PCR was applied to increase specificity and amplicon yield,. The full-length 16S rRNA gene was first amplified with primers 27F and 1492R on 10 ng of template (94 °C—30 s; 50 °C—45 s; and 68 °C—90 s; 30 cycles) using a high-fidelity Pfx polymerase (Life Technologies). PCR products were subsequently purified using the QIAquick PCR purification kit (Qiagen). To obtain dual-index amplicons of the V4 region, a second amplification was performed on 5 μL of PCR product using primers 515F and a slightly modified version of primer 806R to increase detection of SAR11 bacterioplankton for 30 cycles (94 °C—30 s; 55 °C—30 s; and 68 °C—60 s). Both primers contained an Illumina adapter and an 8-nucleotide (nt) barcode at the 5′-end. For each sample, PCRs were performed in triplicate, pooled, and gel-purified using the QIAquick gel extraction kit (Qiagen). An equimolar library was prepared by normalizing amplicon concentrations with a SequalPrep Normalization Plate (Applied Biosystems) and subsequent pooling. Amplicons were sequenced using a v2 PE500 kit with custom primers on the Illumina Miseq platform (KU Leuven Genomics Core), producing 2 × 250-nt paired-end reads. In this way, we generated 24 samples representing 4 genotypes × 2 diets × 3 replicates.Processing of sequencing data: Sequence reads were processed using R 3.3.2 (R Core Team, 2016) following Callahan et al.. Sequences were trimmed (the first 10 nucleotides and from position 180 onwards) and filtered (maximum of 2 expected errors per read) on paired ends jointly. Sequence variants were inferred using the high-resolution DADA2 method, which relies on a parameterized model of substitution errors to distinguish sequencing errors from real biological variation. Chimeras were subsequently removed from the data set. After filtering, the average number of reads per sample was 37,549 (minimum = 14,557 reads and maximum = 66,460 reads). Taxonomy was assigned with a naive Bayesian classifier using the RDP v14 training set. OTUs with no taxonomic assignment at phylum level or which were assigned as “chloroplast” or “cyanobacteria” were subsequently removed from the data set. The final data set contained a total of 894,270 reads, with on average 37,261 reads per sample (minimum = 14,504 reads and maximum = 66,453 reads).Analysis of sequencing data: As measures for α-diversity within the different microbial communities, OTU richness (total number of OTUs present) and Shannon index (taking into account both OTU richness and the relative abundance of OTUs) were calculated using the vegan package in R. The effects of diet (i.e., toxic cyanobacteria vs. nontoxic green algae), tolerance profile (i.e., tolerant vs. susceptible), and genotype (nested within tolerance profile) on OTU richness and Shannon index were assessed through ANOVA. Interactions between the different factors were included in the initial model, and removed when nonsignificant (p > 0.05).To investigate differences in community composition (β-diversity) between the different microbial communities, weighted UniFrac distances were calculated and plotted using principal coordinates analysis with the phyloseq package in R. The effects of diet, tolerance profile, genotype (nested within tolerance profile), and their interactions on β-diversity was assessed through a permutation MANOVA, using the Adonis function of the vegan package in R.To identify the bacterial families that differed between tolerant and susceptible genotypes, OTUs were grouped at the family level, and families representing <1% of the reads were discarded. Differential abundance analyses were then performed with the Bioconductor package DESeq2. […]

Pipeline specifications

Software tools DADA2, UniFrac, phyloseq, DESeq2
Application 16S rRNA-seq analysis
Organisms Daphnia magna, Homo sapiens
Diseases Bloom Syndrome, Drug-Related Side Effects and Adverse Reactions