Pyrosequencing Roche 454 Titanium FLX Approximately 790,000 DNA-enriched beads were loaded into each of 7 quarter regions of two GS Titanium FLX pico titer plates (two separate runs) for sequencing of amplicons and WGS DNA on the Roche 454 GS Titanium FLX platform according to the manufacturer’s specifications. Sequence pre-processing Sequences were processed and split by multiplex identifiers (MIDs) using the sff tools
from Roche 454 of Roche Diagnostics Corp. (Indianapolis, IN). Fusion primer sequences detected on the 5’ and 3’ end of sequences were trimmed. Bioinformatic analyses: 16S rRNA gene analyses The Data Intensive Academic Grid (DIAG) computational cloud (http://diagcomputing.org) was used in combination with the CloVR-16S automated pipeline (Version1.1) [11] to perform computationally-intensive tasks, such as chimera detection and nonparametric statistical learn more analyses, on the 16S rRNA gene sequences. The CloVR-16S pipeline utilizes tools for phylogenetic analysis of 16S rRNA data from Qiime [12] and Mothur [13] for sequence processing and diversity analysis, the RDP Bayesian classifier [14] for taxonomic assignment, UCHIME [15] for chimera mTOR inhibitor detection and
removal, Metastats [7] for statistical comparisons of sample groups, and various R programs for visualization and unsupervised clustering. A full description of the CloVR-16S standard operating procedure (SOP) is available online at http://clovr.org. Phylogenetic analyses of putative Salmonella 16S rRNA gene sequences We used the approximately-maximum-likelihood method for phylogenetic inference implemented in FastTree [16] to further explore the taxonomic identity of Enterobacteriaceae DOK2 sequences
from the different regions of tomato plants. Reference sequences from Enterobacteriaceae and other phyla observed in the samples were used with Salmonella reference sequences from NCBI (Additional file 2: Table S2). Inference was performed using the default settings. Clustering of individuals using the program STRUCTURE [17, 18] was performed with K = 2, and K = 3. Bioinformatic analyses: 18S rRNA gene analysis Sequences were clustered stringently using the Qiime UCLUST module set for a 99% identity threshold. Representatives of each cluster (i.e., the longest read in each cluster) were examined for chimeras using UCHIME [15] in de novo mode. Clusters identified as chimeras were removed from further analysis. Remaining representatives were searched against the SILVA rRNA small subunit (SSU) [19] database (limited to reference sequences with full taxonomic identification) with BLASTN and a minimum e-value threshold of 1e-5. To provide information about overall fungal distribution, the closest known neighbor for each 99% identity cluster was assigned to the taxonomy of the best-BLAST-hit to the representative sequence.