16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae

Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these “conventionalized” larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled “Microbial modulation of behavior and stress responses in zebrafish larvae” (Davis et al., 2016) [1].

More specific subject area Type of data Value of the data The data presented here can be used as justification for the use of zebrafish larvae as a model species in gnotobiotic research.
These data are valuable in illustrating the consistency of microbial taxa present within a given group of larvae.
These data will be of use in the selection of an appropriate methodology to generate gnotobiotic zebrafish larvae.

Data
Data presented here represent results of 16S rRNA sequencing of V4 region amplicons, generated using the Illumina MiSeq platform. Data are presented at the taxonomic levels of phylum, family, and operational taxonomic unit, and represent an average coverage of 4235 reads per sample (Table 1). This paper contains data related to the research concurrently published in Davis et al. [1].

Production of conventionalized and conventionally-raised zebrafish larvae
Wild-type zebrafish breeders were placed into a breeding tank overnight to spawn. Embryos were collected immediately after fertilization and evenly divided into separate groups for subsequent treatment. Conventionalized (CV) embryos were generated by following a previously published method [2]. Briefly, embryos were collected in sterile fish water containing 250 mg/mL amphotericin B, 5 mg/mL kanamycin, and 100 mg/mL ampicillin (AB-fish water). After sorting to remove unfertilized embryos, viable embryos were transferred to a tissue culture hood and gently washed 3 times in ABfish water. Embryos were immersed in 0.1% PVP-Iodine solution for 2 min, and then immediately washed 3 times with sterile fish water. After washing, the embryos were immersed in 0.003% bleach solution for 1 h before being washed an additional 3 times with sterile fish water. Finally, the embryos were transferred into sterile tissue culture flasks containing conventional fish water. Conventionally raised (CR) embryos were transferred and maintained in conventional fish water immediately after collection without undergoing the sterilization process. All zebrafish embryos were maintained in a Table 1 Operational taxonomic units detected in 6 dpf conventionalized and conventionally-raised zebrafish larvae.

Conventionalized
Conventionally-raised 28.5°C incubator and raised at a density of $ 1 embryo/mL until larval stage at 6 days postfertilization (dpf).

Microbial DNA extraction and quantification
Microbial DNA was extracted according to a modified previously published protocol [3]. Immediately following euthanasia, 12 zebrafish larvae were aseptically collected into 800 mL of lysis buffer (500 mM NaCl, 50 mM Tris-HCl, 50 mM EDTA, and 4% SDS), homogenized for 3 min in a Qiagen Tissuelyser II, and incubated at 70°C for 20 min. Following centrifugation at 5000 Â g for 5 min at room temperature, the supernatant was mixed with 200 mL of 10 mM ammonium acetate, incubated on ice for 5 min, and then centrifuged at 16,000 Â g for 10 min at room temperature. 750 mL of supernatant was then mixed with an equal volume of chilled isopropanol, and incubated for 30 min on ice. The contents of the tube were then centrifuged at 16,000 Â g at 4°C for 15 min to pellet DNA. The pellet was rinsed twice with 70% EtOH and re-suspended in 150 mL of tris-EDTA. 15 mL of proteinase-K and 200 mL of buffer AL (DNeasy kit, Qiagen, Valencia, CA) were then added and tubes were incubated at 70°C for 10 min. 200 mL of 100% EtOH was then added and the entire contents of the tube were transferred to a Qiagen spin column before continuing with the manufacturer's instructions for DNA purification (DNeasy Kit, Qiagen). DNA was eluted in 50 mL of EB buffer (Qiagen). Yield of double-stranded DNA was determined via fluorometry (Qubit 2.0, Life Technologies, Carlsbad, CA) using Qubit s dsDNA BR assay kits (Life Technologies).

Metagenomic library preparation and sequencing
Sequencing of the V4 region of the 16S rRNA gene was performed on the Illumina MiSeq platform. Bacterial 16S rRNA amplicons were constructed by amplification of the V4 hypervariable region of the 16S rRNA gene with single-indexed primers flanked by Illumina standard adapter sequences. Universal primers (U515F/806R) previously developed against the V4 region were used for generating amplicons. Oligonucleotide sequences were obtained at proBase. A single forward primer and reverse primers with unique 12-base indices were used in all reactions. PCR reactions (50 mL) contained 100 ng of genomic DNA, forward and reverse primers (0.2 mM each), dNTPs (200 mM each), and Phusion High-Fidelity DNA Polymerase (1U). PCR amplification was performed as follows: amplification at 98°C for 3 min, and 25 cycles at 98°C for denaturation for 15 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, then a final extension at 72°C for 7 min. Amplified product (5 mL) from each reaction was combined and thoroughly mixed; pooled amplicons were purified by addition of Axygen AxyPrep MagPCR Clean-up beads (50 mL) to an equal volume of 50 mL of amplicons and incubated at room temperature for 15 min. Products were washed multiple times with 80% EtOH and the dried pellet resuspended in Qiagen EB Buffer (32.5 mL), incubated at room temperature for 2 min, and then placed on a magnetic stand for 5 min. Supernatant (30 mL) was transferred to a low-binding microcentrifuge tube for storage. The final amplicon pool was evaluated using the Advanced Analytical Fragment Analyzer automated electrophoresis system, quantified with the Qubit flourometer using the quant-iT HS dsDNA reagent kit, and diluted according to the manufacturer's protocol.

Bioinformatics analysis
Assembly, binning, and annotation of DNA sequences were performed at the MU Informatics Research Core Facility (IRCF, Columbia, MO). Briefly, contiguous sequences of DNA were assembled using FLASH software [4] and contigs were culled if found to be short after trimming for a base quality less than 31. Qiime v1.7 [5] software was used to perform de novo and reference-based chimera detection and removal, and remaining contigs were assigned to operational taxonomic units (OTUs) using a criterion of 97% nucleotide identity. Taxonomy was assigned to selected OTUs using BLAST [6] against the Greengenes database [7] of 16S rRNA sequences and taxonomy.