Computational protocol: Comparison of microbial taxonomic and functional shift pattern along contamination gradient

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[…] DNA was extracted using a TIANamp Bacterial DNA Kit (MO BIO Laboratories, Inc., Carlsbad, CA). The V4 region of the 16S rRNA genes was amplified with the primer pair 515 F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’- GGACTACHVGGGTWTCTAAT-3’). Sample libraries were generated from purified PCR products. The MiSeq 500 cycles kit was used for 2x250 bp paired-ends sequencing on MiSeq machine (Illumina, San Diego, CA). Sequences with perfect matches to barcodes were split to sample libraries, and trimmed. OTU clustering was performed through UCLUST at 97 % similarity level [], and taxonomic assignment was through the RDP classifier [] with a minimal 50 % confidence estimate. The above steps were conducted through the Galaxy pipeline ( developed by Qin el al. Subsequent analyses were performed in R []. Finally, samples were rarefied at 13,000 sequences per sample. All the 16S rRNA sequences were deposited in GenBank database and the accession number were KP784842 - KP788032.For each sample, microbial community DNA was extracted and purified as described previously [, ]. Amplified DNA was labeled and hybridized with GeoChip 5.0, which is a powerful tool to study the functional diversity, composition, structure and metabolic potential of microbial communities []. All GeoChip 5.0 hybridization data are available at the Institute for Environmental Genomics, University of Oklahoma ( The hybridized GeoChip 5.0 was analyzed as previously described []. Software TMEV was used for hierarchical cluster analysis of sequencing and GeoChip data. Statistical differences between the functional microbial communities from the different sites were analyzed by analysis of variance (ANOVA). […]

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