README file for Smith et al



The MIAME information for this experiment is divided into the standard six sections:



1. The raw hybridization data for each sample is in Affymetrix CEL file format.



2. The final processed hybridization data set used to draw conclusions from the study.  Data have been formatted as three separate files.



Smith et al_MIAME_Phylochip bacterial abundance data.xls (OTU ID and Phylochip HybScores)



Smith et al_MIAME_Phylochip bacterial binary data.xls (OTU ID and Phylochip presence-absence data)



Smith et al_MIAME_Phylochip archaeal binary data.xls (OTU ID and Phylochip presence-absence data)



3. The essential sample annotations including experimental categories.



Smith et al_MIAME_metadata.xls (Sample manifest and experimental categories)



Smith et al_MIAME_Phylochip OTU annotations.xls



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).



Same files as listed above.



5. Reference commercial array catalog number: Affymetrix GeneChip: 520660



6. Essential laboratory and data processing protocols.



PhyloChip™ microarrays are commercially available through Second Genome, Inc. (San Bruno, CA) and have been described extensively (technical details, quality/processing controls, and reproducibility tests) by Hazen et al (Science 2010).  Oligonucleotide targets and probes on PhyloChip™ version G3 were synthesized by photolithography, representing publicly available bacteria and archaea 16S rRNA genes.  Operational taxonomic units (OTUs) were defined as a group of highly similar 16S rRNA gene sequences (< 0.5% divergence).  In total, there were 59,959 potential clusters, spanning 2 domains (archaea and bacteria), 147 phyla, 1,123 classes, and 1, 219 orders.  Each OTU was assigned to one of 1,464 families according to methods outlined by Hugenholtz (Genome Biol 2002).  



Amplified and purified DNA products were fragmented and biotin labeled using commercial kits from Affymetrix (Santa Clara, CA).  As detailed by Hazen et al. (Science 2010): fragmented 16S amplicons and non-16S quantitative amplicon reference controls were labeled with biotin in 40 ?L reactions containing: 8 ?L 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 ?L of 0.5M EDTA was added to terminate the reaction. Labeled DNA was combined with 65 ?L of 2X MES hybridization buffer, 20.4 ?L of DMSO, 2 ?L of Affymetrix control oligo B2, and 0.4 ?L nuclease free water. Each reaction mixture was injected into the hybridization chamber of version G3 of a PhyloChip™ Array and incubated for 16 h at 48°C and 60 RPM. Then the PhyloChip™ was washed, stained, and scanned using a GeneChip Scanner 3000 7G (Affymetrix).  Fluorescence intensity was captured using Affymetrix software (GeneChip® Microarray Analysis Suite). 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. 



