README file for Dubinsky_2012_ES&T


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. 

2. The final processed data for the set of hybridizations in the study formatted as a data matrix used to draw the conclusions from the study. 
MIAME_PhyloChip_data.xlsx 

3. The essential sample annotation including experimental factors. 
MIAME_metadata.xlsx

4. The experimental design including sample data relationships (e.g., which raw data file relates to which sample, which hybridizations are technical, which are biological replicates). 
MIAME_metadata.xlsx 

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

6. Essential laboratory and data processing protocols. 
From isolated gDNA extracted from fecal samples and water filters, 500 ng of bacterial PCR product was prepared for PhyloChip hybridization. PCR products were fragmented according to the previously described method (Brodie et al. AEM 2006) to a range of 50-200 bp as verified by agarose gels. Commercial kits were utilized for DNA labeling (Affymetrix WT Double Stranded DNA Terminal Labeling), and Affymetrix GeneChip Hybridization, Wash, and Stain kits were used for PhyloChip processing. Briefly, fragmented 16S amplicons and non-16S quantitative amplicon reference controls were labeled with biotin in 40 μL reactions containing: 8 μL of 5X TDF buffer, 40 units of TDF, and 3.32 nanomoles of GeneChip labeling reagent. After incubating at 37°C for 60 min, 2 μL of 0.5M EDTA was added to terminate the reaction. Labeled DNA was combined with 65 μL of 2X MES hybridization buffer, 20.4 μL of DMSO, 2 μL of Affymetrix control oligo B2, and 0.4 μL nuclease free water. Each reaction mixture was injected into the hybridization chamber of an array cartridge and incubated for 16 h in an Affymetrix hybridization oven at 48°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 occupied approximately 8x8 pixels in the image file corresponding to 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.