Computational protocol: Rapid Formation of Microbe Oil Aggregates and Changes in Community Composition in Coastal Surface Water Following Exposure to Oil and the Dispersant Corexit

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Protocol publication

[…] Total DNA was extracted from filters using FastDNA Spin kits (MP Biomedical; Santa Ana, California) and stored at −20°C until PCR amplification. Three sample-free filters were processed as protocol blanks in order to control for the possible presence of DNA contamination in the extraction kits and PCR reagents (Salter et al., ). The hyper-variable V4 region of the 16S rRNA gene was PCR amplified from the DNA extracts with GoTaq Flexi DNA Polymerase (Promega, Fitchburg, USA) using a methodology similar to that described by Caporaso et al. (). Each sample was amplified in triplicate 25 μL reactions with the following cycling parameters: 95°C for 3 min, 30 cycles of 95°C for 45 s, 50°C for 60 s, and 72°C for 90 s, and a final elongation step at 72°C for 10 min. All amplifications were performed using the 515F-806R primer pair (10 μM each) modified to include recently published revisions that reduce bias against the Crenarchaeota and Thaumarchaeota lineages as well as the SAR11 bacterial clade (Apprill et al., ; Parada et al., ). The primer pair was additionally modified to include Golay barcodes and adapters for Illumina MiSeq sequencing. Final primer sequences are detailed in Walters et al. (). Following amplification, the triplicate products were combined together and run on a 1.5% agarose gel to assess amplification success and relative band intensity. Amplicons were then quantified with the QuantiFluor dsDNA System (Promega), pooled at equimolar concentrations, and purified with an UltraClean PCR Clean-Up Kit (MoBio Laboratories; Carlsbad, USA). The purified library, along with aliquots of the three sequencing primers, were sent to the Georgia Genomics Facility (Athens, GA, USA) for MiSeq sequencing (v2 chemistry, 2 × 250 bp).Primers P450fw1 (GTSGGCGGCAACGACACSAC) and P450rv3 (GCASCGGTGGATGCCGAAGCCRAA) were used to obtain PCR products for cytochrome P450 alkane hydroxylase (van Beilen et al., ). PCR products were ligated into competent E. coli cells and cloned using the TOPO TA Cloning Kit (Thermo Fisher Scientific; Waltham, USA). Plasmid extractions were performed using Qiagen Mini-prep Kits. Sequencing of seven random clones verified the success of the cloning reaction and exact size and sequence of the targeted PCR products were confirmed with BlastX. A mixture of five P450 clone sequences were used to create the standard, three Alphaproteobacteria and one Gammaproteobacteria. qPCR was carried out for all samples in triplicate 25 μL reactions using 12.5 μL of iQ SYBR Green Supermix (Bio-Rad; Hercules, USA) and 10 μM of each primer with the following cycling parameters: 95°C for 3 min, 40 cycles of 95°C for 10 s, 57°C for 30 s, and 72°C for 45 s, and a final elongation step at 72°C for 45 s. A melt-curve was included at the end of each PCR cycle to confirm only a single product was amplified. Sequences of these standards are available on GenBank under accession numbers MF962916-MF962919. [...] Sequence read curation and processing were carried out using mothur v.1.36.1 (Schloss et al., ) following a modified version of the protocol described in Kozich et al. (). Paired reads were combined into contigs and quality filtered to exclude sequences containing ambiguous base calls or homopolymer runs longer than 8 bp. The merged contigs were then aligned with the SILVA non-redundant 16S rRNA reference dataset (v.123). To help mitigate the generation of spurious sequences, the aligned sequences were “pre-clustered,” allowing 1-bp difference per 100 bp of sequence (Huse et al., ). Chimeric sequences were identified with the UCHIME algorithm (Edgar et al., ) and removed from further processing and analysis. Sequences were then clustered into OTUs at 3% or less dissimilarity using the average neighbor method (Schloss and Westcott, ). As a final quality control measure, potential contaminant sequences were screened by removing any OTU from the dataset wherein 1% or greater of its sequences were sourced from any of the three protocol blank libraries.We constructed 10,079,362 contigs with an average read length of 255 bp distributed across 87 barcoded samples from Illumina MiSeq paired-end 250-bp sequencing. After screening of sequences with ambiguous bases or long homopolymers, 694,269 unique sequences were used to generate an alignment. Further quality control measures (i.e., pre-clustering, alignment curation, and chimera removal) reduced the total library to 138,542 unique contigs which clustered into 9,920 OTUs. As a final curation step, OTUs in which 1% or greater of its member sequences were from a protocol blank library were considered likely background contamination and removed from the dataset. Twenty-six OTUs met this criteria for removal, resulting in a final curated dataset containing 9894 OTUs clustered from 6,230,128 sequences. Each library contained an average of 74,168 ± 18,927 sequences (Table ). Rarefaction curves for all samples approached saturation (Figure ), and together with high Good's Coverage values (>99%, Table ) indicate that the remaining un-sampled diversity likely contained only rare taxa.A consensus taxonomy for each OTU was assigned using a naïve Bayesian classifier (Wang et al., ) trained on the SILVA reference database (Quast et al., ) with a confidence threshold of 80%. The taxonomic assignments for OTUs of interest were then checked by classifying a representative sequence, selected as the most abundant within the OTU, with the Greengenes, RDP, SILVA, GenBank, and EZ-Taxon databases (Table ). Diversity and richness indices (observed richness, Inverse Simpson, Chao1, ACE) were calculated from the average (1000 iterations) of rarefying libraries to the smallest library size (23,242 sequences). Patterns in microbial community structure were examined using non-metric multidimensional scaling (NMDS) based on the Bray-Curtis dissimilarity index (Bray and Curtis, ), and tested for significance using analysis of molecular variance, AMOVA (Excoffier et al., ). […]

Pipeline specifications

Software tools BLASTX, mothur, UCHIME
Application 16S rRNA-seq analysis
Organisms Escherichia coli, Bacteria