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].

Specifications Table

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].

Table 1

Operational taxonomic units detected in 6 dpf conventionalized and conventionally-raised zebrafish larvae.

			Conventionalized			Conventionally-raised
Phylum	Family	Operational taxonomic unit	Mean (%)	SEM (%)	Prevelance (%)	Mean (%)	SEM (%)	Prevelance (%)
		No blast hit;Other	2.69	0.39	100	2.22	0.19	100
Actinobacteria	Microbacteriaceae	Family Microbacteriaceae	0.00	0.00	0	0.00	0.00	25
	Mycobacteriaceae	Mycobacterium sp.	0.00	0.00	0	0.02	0.01	100
	Bifidobacteriaceae	Bifidobacterium sp.	0.02	0.02	25	0.00	0.00	0
	unclassified	Order Solirubrobacterales	1.82	0.19	100	1.08	0.05	100
Bacteroidetes	unclassified	Order Bacteroidales	0.46	0.16	100	0.13	0.03	100
	Bacteroidaceae	Bacteroides sp.	0.29	0.26	50	0.31	0.12	100
		Bacteroides acidifaciens	0.03	0.03	25	0.01	0.01	50
	Porphyromonadaceae	Parabacteroides sp.	0.25	0.22	50	0.04	0.02	75
	Prevotellaceae	Family Prevotellaceae	0.05	0.03	50	0.06	0.04	50
		Prevotella sp.	0.00	0.00	0	0.01	0.01	25
	Rikenellaceae	Family Rikenellaceae	0.15	0.06	75	0.02	0.01	75
	S24-7	Family S24-7	1.10	0.91	100	0.14	0.05	100
	Barnesiellaceae	Family Barnesiellaceae	0.02	0.02	25	0.01	0.00	50
	Paraprevotellaceae	YRC22 sp.	0.00	0.00	0	0.01	0.01	25
	Cytophagaceae	Family Cytophagaceae	0.07	0.04	50	5.05	0.09	100
		Emticicia sp.	0.00	0.00	0	0.13	0.02	100
		Flectobacillus sp.	0.00	0.00	0	3.19	0.13	100
		Hymenobacter sp.	0.00	0.00	0	0.72	0.05	100
		Runella sp.	0.00	0.00	0	2.22	0.12	100
		Spirosoma sp.	0.00	0.00	0	0.20	0.03	100
	Cryomorphaceae	Fluviicola sp.	0.00	0.00	0	0.05	0.01	100
	Flavobacteriaceae	Flavobacterium sp.	0.03	0.03	25	1.05	0.06	100
		Flavobacterium columnare	0.08	0.03	75	0.01	0.01	25
	Weeksellaceae	Chryseobacterium sp.	0.00	0.00	0	0.63	0.10	100
	unclassified	Order Sphingobacteriales	0.15	0.06	75	2.28	0.12	100
	Sphingobacteriaceae	Pedobacter sp.	0.21	0.07	100	1.20	0.09	100
		Sphingobacterium multivorum	0.00	0.00	0	17.50	0.53	100
	Chitinophagaceae	Family Chitinophagaceae	0.00	0.00	0	0.04	0.02	100
		Sediminibacterium sp.	0.06	0.03	50	0.01	0.01	25
	Saprospiraceae	Family Saprospiraceae	0.00	0.00	0	0.71	0.07	100
		Saprospira sp.	0.00	0.00	0	0.14	0.03	100
Chloroflexi	SHA-31	Family SHA-31	0.00	0.00	0	0.11	0.01	100
Cyanobacteria	unclassified	Order YS2	0.02	0.02	25	0.00	0.00	0
		Order Stramenopiles	12.56	1.22	100	8.96	0.49	100
Deferribacteres	Deferribacteraceae	Mucispirillum schaedleri	0.05	0.05	25	0.01	0.01	25
Firmicutes	Staphylococcaceae	Staphylococcus succinus	0.03	0.03	25	0.00	0.00	0
	Lactobacillaceae	Lactobacillus sp.	0.03	0.03	25	0.01	0.01	25
	Turicibacteraceae	Turicibacter sp.	0.00	0.00	0	0.00	0.00	25
	unclassified	Order Clostridiales	0.31	0.20	75	0.27	0.05	100
	Clostridiaceae	Family Clostridiaceae	0.02	0.02	25	0.00	0.00	0
		Clostridium sp.	0.10	0.10	25	0.04	0.02	50
	Dehalobacteriaceae	Dehalobacterium sp.	0.03	0.03	25	0.00	0.00	25
	Lachnospiraceae	Family Lachnospiraceae	0.10	0.04	75	0.07	0.01	100
		Coprococcus sp.	0.04	0.04	25	0.02	0.01	50
		Coprococcus eutactus	0.00	0.00	0	0.00	0.00	25
		Roseburia sp.	0.00	0.00	0	0.01	0.01	50
		Ruminococcus gnavus	0.00	0.00	0	0.00	0.00	25
	Peptococcaceae	Family Peptococcaceae	0.03	0.03	25	0.00	0.00	0
		rc4-4 sp.	0.02	0.02	25	0.00	0.00	25
	Peptostreptococcaceae	Family Peptostreptococcaceae	0.02	0.02	25	0.00	0.00	0
	Ruminococcaceae	Family Ruminococcaceae	0.18	0.07	75	0.19	0.04	100
		Oscillospira sp.	0.25	0.09	75	0.08	0.02	100
		Ruminococcus sp.	0.00	0.00	0	0.03	0.01	75
		Ruminococcus flavefaciens	0.05	0.03	50	0.00	0.00	0
	Erysipelotrichaceae	Family Erysipelotrichaceae	0.04	0.04	25	0.00	0.00	0
		Allobaculum sp.	0.09	0.09	25	0.00	0.00	25
Proteobacteria	Caulobacteraceae	Family Caulobacteraceae	1.15	0.25	100	0.14	0.01	100
		Asticcacaulis sp.	0.00	0.00	0	0.19	0.02	100
	unclassified	Order RF32	0.14	0.14	25	0.01	0.00	50
		Order Rhizobiales	0.02	0.02	25	0.02	0.00	100
	Aurantimonadaceae	Family Aurantimonadaceae	0.00	0.00	0	0.08	0.01	100
	Bradyrhizobiaceae	Bosea genosp.	0.03	0.03	25	0.01	0.01	50
	Hyphomicrobiaceae	Hyphomicrobium sp.	0.02	0.02	25	0.03	0.01	75
	Phyllobacteriaceae	Family Phyllobacteriaceae	0.03	0.03	25	0.04	0.01	100
	Rhizobiaceae	Agrobacterium sp.	0.09	0.06	50	0.05	0.01	100
	Hyphomonadaceae	Family Hyphomonadaceae	0.00	0.00	0	0.16	0.02	100
	Rhodobacteraceae	Paracoccus aminovorans	0.00	0.00	0	0.02	0.01	50
		Rhodobacter sp.	0.03	0.03	25	0.01	0.01	25
	Rhodospirillaceae	Family Rhodospirillaceae	0.00	0.00	0	0.11	0.01	100
		Phaeospirillum fulvum	0.00	0.00	0	0.07	0.02	100
	unclassified	Order Rickettsiales	1.00	0.14	100	0.73	0.10	100
	Rickettsiaceae	Family Rickettsiaceae	0.22	0.05	100	0.36	0.04	100
	mitochondria	Vermamoeba vermiformis	0.00	0.00	0	0.01	0.01	50
	Sphingomonadaceae	Novosphingobium sp.	0.00	0.00	0	0.00	0.00	25
		Sphingomonas sp.	0.30	0.06	100	0.06	0.03	100
		Sphingomonas yabuuchiae	0.00	0.00	0	0.16	0.04	100
		Class Betaproteobacteria	0.14	0.06	75	0.10	0.03	100
	Alcaligenaceae	Sutterella sp.	0.08	0.08	25	0.04	0.01	100
	Comamonadaceae	Family Comamonadaceae	2.21	0.19	100	22.21	0.38	100
		Comamonas sp.	0.05	0.03	50	0.15	0.02	100
		Limnohabitans sp.	1.77	0.42	100	6.53	0.08	100
		Variovorax paradoxus	0.13	0.05	75	0.07	0.03	100
	Oxalobacteraceae	Family Oxalobacteraceae	0.00	0.00	0	0.19	0.02	100
		Janthinobacterium sp.	0.00	0.00	0	0.01	0.01	25
	Methylophilaceae	Methylotenera mobilis	0.13	0.01	100	0.02	0.01	75
	Rhodocyclaceae	Family Rhodocyclaceae	4.47	0.26	100	3.16	0.13	100
	Bdellovibrionaceae	Bdellovibrio sp.	0.00	0.00	0	0.25	0.03	100
		Bdellovibrio bacteriovorus	1.59	0.13	100	0.43	0.03	100
	unclassified	Order Myxococcales	0.00	0.00	0	0.03	0.01	75
	Helicobacteraceae	Family Helicobacteraceae	0.03	0.03	25	0.00	0.00	0
	Alteromonadaceae	Cellvibrio sp.	0.00	0.00	0	2.83	0.18	100
	Chromatiaceae	Rheinheimera sp.	61.49	1.07	100	8.57	0.36	100
	Coxiellaceae	Family Coxiellaceae	0.00	0.00	0	0.01	0.01	50
	Legionellaceae	Legionella sp.	0.08	0.03	75	0.01	0.01	50
	Moraxellaceae	Family Moraxellaceae	0.00	0.00	0	0.03	0.01	100
	Pseudomonadaceae	Pseudomonas sp.	0.40	0.14	100	0.00	0.00	0
		Pseudomonas pseudoalcaligenes	0.00	0.00	0	0.04	0.00	100
	Sinobacteraceae	Family Sinobacteraceae	1.83	0.26	100	0.69	0.05	100
		Nevskia ramosa	0.83	0.17	100	2.71	0.12	100
	Xanthomonadaceae	Stenotrophomonas sp.	0.02	0.02	25	0.01	0.00	50
Spirochaetes	Spirochaetaceae	Treponema sp.	0.00	0.00	0	0.01	0.01	25
Synergistetes	Dethiosulfovibrionaceae	Family Dethiosulfovibrionaceae	0.00	0.00	0	0.01	0.01	25
TM6	unclassified	Class SBRH58	0.04	0.04	25	0.30	0.04	100
TM7	F16	Family F16	0.00	0.00	0	0.00	0.00	25
Tenericutes	Anaeroplasmataceae	Anaeroplasma sp.	0.00	0.00	0	0.00	0.00	25
	unclassified	Order RF39	0.00	0.00	0	0.02	0.00	100
Verrucomicrobia	unclassified	Order HA64	0.00	0.00	0	0.01	0.01	25
	Opitutaceae	Family Opitutaceae	0.00	0.00	0	0.09	0.02	100
	RFP12	Family RFP12	0.00	0.00	0	0.02	0.02	25
	Verrucomicrobiaceae	Akkermansia muciniphila	0.20	0.20	25	0.09	0.05	75
Thermi	Deinococcaceae	Deinococcus sp.	0.03	0.03	25	0.11	0.02	100

Experimental design, materials and methods

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 µg/mL kanamycin, and 100 µg/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 AB-fish 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 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 µL 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× g for 5 min at room temperature, the supernatant was mixed with 200 µL of 10 mM ammonium acetate, incubated on ice for 5 min, and then centrifuged at 16,000× g for 10 min at room temperature. 750 µL 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× g at 4 °C for 15 min to pellet DNA. The pellet was rinsed twice with 70% EtOH and re-suspended in 150 µL of tris-EDTA. 15 µL of proteinase-K and 200 µL of buffer AL (DNeasy kit, Qiagen, Valencia, CA) were then added and tubes were incubated at 70 °C for 10 min. 200 µL 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 µL of EB buffer (Qiagen). Yield of double-stranded DNA was determined via fluorometry (Qubit 2.0, Life Technologies, Carlsbad, CA) using Qubit® 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 µL) contained 100 ng of genomic DNA, forward and reverse primers (0.2 µM each), dNTPs (200 µM 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 µL) from each reaction was combined and thoroughly mixed; pooled amplicons were purified by addition of Axygen AxyPrep MagPCR Clean-up beads (50 µL) to an equal volume of 50 µL 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 µL), incubated at room temperature for 2 min, and then placed on a magnetic stand for 5 min. Supernatant (30 µL) 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.

Subject area	Biology
More specific subject area	Microbiome analysis in zebrafish larvae
Type of data	Table
How data was acquired	Illumina MiSeq
Data format	Raw, analyzed
Experimental factors	Reconstitution of sterilized embryos with conventional microbial populations
Experimental features	1) Microbial DNA extraction and amplification via PCR
	2) Bacterial 16S rRNA amplicon sequencing
	3) Trimming, filtering, and annotation of sequence data
Data source location	Columbia, MO, USA
	Latitude: 38.901366 Longitude: −92.2825 Altitude: 246 m
Data accessibility	Data is within this article and available via http://www.ncbi.nlm.nih.gov/bioproject/321905