Computational protocol: Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables

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Protocol publication

[…] Following homogenization of each sample, a subsample of the slurry (50 mL) was filtered through a sterile 11 Whatman 1 filter (11 μm nominal pore size) to remove residual leaf particles. 35 mL of the filtrate was collected and centrifuged (8,000 × g, 10 min) to pellet cells, and the moist pellet transferred to a 1.5 ml sterile microcentrifuge tube. This pellet was further centrifuged (8,000 × g, 10 min), the supernatant removed, and the pellet frozen at −20°C until DNA extraction. DNA was extracted using a PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) and a fragment of the bacterial 16S rRNA gene amplified using Bac799f (5’-AACMGGATTAGATACCCKG-3’) and Univ1492r (5’-GGTTACCTTGTTACGACTT-3’) primers. This combination of primers targets bacterial DNA specifically without amplifying residual chloroplast DNA from the host plant. Plant mitochondrial DNA is co-amplified, but yields a 1,090 bp fragment compared to a 735 bp fragment for bacterial DNA [-]. PCR was carried out in 50 μl reactions following procedures described previously []. Amplification products were visualized on 1% agarose gels, which also separated bacterial and host plant mitochondrial DNA fragments. The bacterial gel band was excised and DNA recovered from the gel fragments using UltraClean GelSpin DNA Extraction Kits (Mo Bio Laboratories, Carlsbad, CA). These purified bacterial 16S rRNA gene fragments were used as the templates for pyrosequencing. Negative control amplifications (no template DNA) were carried out routinely and yielded no detectable product.Bacterial tag-encoded FLX amplicon 454 pyrosequencing (bTEFAP) [] was conducted on the 16S rRNA gene amplicons of each sample, through a dedicated sequencing facility (MR DNA, Shallowater, TX). Bacterial primers 939f and 1392r [,] were used in the sequencing reaction. A single-step PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) was used under the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 sec, 53°C for 40 sec, and 72°C for 1 min, after which a final elongation step at 72°C for 5 min was performed. Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt AMPure XP beads (Agencourt Bioscience Corporation, Danvers, MA). Samples were sequenced utilizing Roche 454 FLX titanium instruments and reagents and following the manufacturer’s guidelines. A negative control amplification was used in the same 454 reaction and gave no valid reads.Raw pyrosequence data derived from the sequencing process was transferred into FASTA files for each sample, along with sequencing quality files. Files were accessed using the bioinformatics software Mothur [] where they were processed and analysed following general procedures recommended by Schloss et al. []. Briefly, sequences were denoised, and trimmed to remove barcodes and primers. Sequences were aligned and classified according to those in the SILVA rRNA database [], after which chimeric sequences and any sequences classified as mitochondria or chloroplasts were removed from the dataset. Remaining sequences were grouped into operational taxonomic units (OTUs) based on a 97% similarity criterion. Rarefaction was performed on each sample to assess sampling adequacy, using a 50 sequence increment. Random subsamples (1000) of OTUs from each sample corresponding to the number of sequences in the lowest sample (i.e. smallest sample size) were then used for further analysis. The same subsampling approach was used to examine variation in community structure between samples (beta diversity) using the theta similarity index of Yue and Clayton, an index that accounts for proportional abundances of both shared and non-shared OTUs []. Similarity between samples was visualized by ordination of samples by non-metric multidimensional scaling (NMDS) as well as dendrogram construction. Spatial separation of samples in NMDS was tested through analysis of molecular variance (AMOVA), while clustering of samples within the dendrogram was tested using the UniFrac distance metric []. […]

Pipeline specifications

Software tools mothur, UniFrac
Application 16S rRNA-seq analysis
Organisms Homo sapiens