README file for Kellogg_2013_PLoS ONE

The MIAME information for this experiment is divided into the standard six sections. 
1. The raw data for each hybridization: 
The raw data from each sample is in Affymetrix CEL file format. The CEL files are for the G3 version of the PhyloChipTM microarray.

2. The final processed data (binary and hybridization intensity data) for the set of samples in the study formatted as a data matrix used to draw the conclusions from the study. 
For the binary data, a value of one indicates a positive call for an OTU in a sample and a zero value indicates a negative call using the criteria stated in Section 6. 
For the intensity data, the values for each sample are trimmed mean (mean without the highest and lowest values) of the probe intensities for the perfectly-matched probes used to define each OTU. These values were scaled to an internal spike mix to account for array-to-array differences in intensity.
MIAME_PhyloChip_data_Orbicella_Kellogg.xlsx 

3. The essential sample annotation including experimental factors: 
MIAME_metadata_Orbicella_Kellogg.xlsx

4. The experimental design including sample data relationships (e.g., which raw data file relates to which sample, disease state, collection location): 
Same file as above. 

5. Reference commercial array catalog number:
G3 Second Genome PhyloChip(TM) Assay: PN49-0002A

6. Essential laboratory and data processing protocols: 
From gDNA extracted from coral tissue, 500 ng of bacterial PCR product was prepared for PhyloChip hybridization. Sample VIISMAH06 had 450 ng loaded due to less robust amplification.  PCR products were fragmented according to the previously described method (using DNAse, Hazen et al, 2010) to a range of 50-200 bp. DNA labeling was as described in Hazen et al. (2010), and Affymetrix GeneChip Hybridization, Wash, and Stain kits were used for PhyloChip processing. Briefly, the fragmented product was biotinylated using 8 µL 5x TdT buffer, 1.34 µL TdT enzyme, and 0.66 µL GeneChip Labeling Reagent in a final volume of 40 µL, incubated at 37 degrees C for one hour, 70 degrees C for 10 min., and then 4 degrees C for 10 min.  Two microliters of 0.5 M EDTA was added to stop all enzymatic reactions.  The product was prepared for hybridization onto the array by adding 2.2 µL Control Oligo B2, 65 µL 2x Hybe Mix, 20.4 µL DMSO, and sufficient water to bring the final volume to 130 µL.  This mixture was incubated at 99 degrees C for 5 min. to denature the DNA strands and then held at 48 degrees C for 5 min. before being transferred to a pre-hybridized array also being held at 48 degrees C.  Each reaction mixture was applied to the hybridization chamber of an array cartridge and incubated for 16 h in an Affymetrix hybridization oven at 48 degrees C and 60 RPM. Hybridization solution was removed and the microarrays were stained according to the manufacturer's instructions. Fluorescent images were captured with the GeneChip Scanner 3000 7G (Affymetrix, Santa Clara, CA). 

An individual array feature was composed of a single probe 25mer on the surface. The central 9 pixels were ranked by intensity and the 75th percentile was used as the summary intensity for the feature. Probe intensities were background-subtracted and scaled to controlled Quantitative Standards (non-16S spike-ins) and outliers were identified as previously described (DeSantis et al. Microbial Ecology 2007). The hybridization score (HybScore) for an OTU was calculated as the mean intensity of the perfectly matching probes exclusive of the maximum and minimum.
For binary data, the following parameters were used (as defined in Hazen et al. (2010): Stage 1 of PhyCA analysis was performed using cutoff values: q1= 0.5, q2=0.93, q3=0.98 . Stage 2 of the analysis was performed using cutoff values: q1=0, q2=0, and q3=0.1.  
[See manuscript for references.]