Computational protocol: Optimization of DNA extraction for advancing coral microbiota investigations

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Protocol publication

[…] Mothur software [] (v.1.33.3) was used to combine the de-multiplexed paired reads (8,344,281 contigs) and remove longer sequences (>275 bp) and sequences containing ambiguous base pairs. The expected length of the amplified region with the PCR-specific primers removed was 254 bp. A subset of longer sequences with read lengths exceeding 275 bp were queried using the NCBI BLASTN 2.3.0 program [, ] to evaluate the taxonomic affiliation of these sequences. The remaining sequences were classified using the SILVA SSU Ref database [] (v. 119), and sequences corresponding to Eukaryota, mitochondria, and “unknown” lineages were discarded (2802 sequences). Chloroplast sequences were retained to assess if more chloroplast sequences were associated with a particular DNA extraction treatment. The UCHIME algorithm [] was used to identify and remove chimeric sequences (13,724 sequences total). Sequences were not subsampled [, ].The sequences were grouped into nodes using the Minimum Entropy Decomposition (MED) algorithm []. These MED nodes are analogous to operational taxonomic units (OTUs) and resolve biologically meaningful and distinct groups that can be separated by <1% sequence disparities [, ]. Taxonomy was assigned to MED nodes using the classify.seqs command in mothur [] and the SILVA database (v. 119) []. Sequences belonging to “unclassified” MED nodes were re-aligned using the SINA alignment service [] (v. 1.2.11) and imported into ARB [] using the SILVA v. 123 database where phylogenetic comparisons were made using neighbor joining algorithms to resolve “unclassified” taxonomy. The mock community and positive control DNA sample yielded the expected communities, replicate barcoded samples produced repeatable results, and the negative control samples did not pass quality control; these samples were then excluded from the analysis. [...] DNA concentrations were tested for normality using the Shapiro-Wilk test. Concentrations were then subjected to one-way analysis of variance (ANOVA) or Friedman repeated measures analysis of variance (FRMANOVA) on ranks tests, if data failed the Shapiro-Wilk test, to assess if there were significant differences in mean DNA concentrations between coral species or DNA extraction methods. When appropriate, Tukey’s, Holm-Sidak, or Dunn’s method post hoc tests were used to determine significantly different groups. p values ≤0.05 were accepted as being statistically significant. The above statistical tests were conducted using SigmaPlot software (v. 13).Primer (v.7.0.9, Primer- E Ltd.) was used for a majority of the microbial community visualization and alpha diversity analysis. MED richness was calculated using the average number of unique MED nodes detected for each species × treatment grouping. MED species evenness was determined using the averaged Pielou’s evenness index (J’). Bray-Curtis distances were calculated from normalized, square-root transformed sequence data and used to conduct non-metric multidimensional scaling (nMDS) and nested two- and one-way analysis of similarity (ANOSIM) tests. Presence/absence heat maps of MED nodes detected from O. faveolata, O. annularis, and A. humilis associated amplicons were created using the phyloseq [] R package and a custom script [] that was modified for this study. These heat maps were generated using distinct MED nodes that comprised 50% of all the reads obtained for each sample and thus represent the most dominant groups found within each colony. Frequency of MED node detection was determined for each treatment (and referred to as the top 50% MED coverage percentage), and the percentage of detection agreement between pairwise treatments within each colony was assessed. One-tailed t tests were used to reveal significantly different MED node detection between treatments and were conducted using SigmaPlot. […]

Pipeline specifications

Software tools mothur, BLASTN, UCHIME, SigmaPlot, phyloseq
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Organisms Orbicella faveolata, Orbicella annularis, Acropora humilis