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Fig. 1
Experimental design. Overview of the methods analyzed for impact on interpretation of experimental results. First panel, 384 zebrafish larvae were distributed across the wells of four 96-well round-bottom plates. For two of these plates, the larvae were left untreated, while the larvae of the other two plates were exposed to 10 µM benzo-[a]-pyrene by way of their growth media. The zebrafish were raised in their wells on these plates until 9 days post fertilization. Second panel, for each of the four plates we conducted a fully factorial splitting of the samples by dissection method and DNA extraction kit. Half of the samples on each plate had their intestines dissected and placed in the collection tube, while for the other half, we placed the whole carcasses in their collection tubes. Then, in a manner orthogonal to dissection method, a third of each set of samples underwent DNA extraction using one of the three extraction kits. Third panel, we prepared 16S rDNA sequencing libraries using either single or triplicate PCR. Every sample was subject to both of these treatments. These libraries were then submitted for sequencing. Bottom panel, upon receiving the 16S sequences, we processed the data for quality and conducted the analyses described herein.