Pipeline biofilm samples
Three samples scraped from the inner surface of oilfield pipelines (i.e. pigged pipeline material) were collected and stored at −80°C. Two of the samples, designated "A" and "B", originated from pipelines carrying produced water being returned to the formation to maintain pressure. A third sample, "C", originated from a pipeline as part of a seawater injection system for secondary oil recovery. Samples A and B contained crude oil, corrosion products such as iron sulfides and biofilm material (e.g. EPS). Sample C did not contain crude oil but did contain lesser quantities of corrosion products and biofilm material. For samples A, B, and C, DNA was extracted from ten aliquots (subsamples) for each extraction platform. For samples A and B (40 ml), each was thawed at 4°C, mixed, and ten replicate subsamples (0.5 ml) were dispensed into 2 ml conical screw-top tubes. Sample C (40 ml) was thawed, mixed, and ten replicate subsamples (1 ml) were dispensed into 2 ml conical tubes and concentrated by centrifugation for 5 min at 14000 × g, removing 0.5 ml of the supernatant and re-suspending the remaining volume. This concentration of biomass was deemed necessary for sample C, as initial studies revealed it contained 1/10th of the biomass of samples A or B (personal communication, Kathleen Duncan).
MOBIO PowerBiofilm extraction platform
The PowerBiofilm DNA Isolation Kit (MOBIO Laboratories) was used to manually extract DNA from ten replicate subsamples of samples A, B, and C according to the manufacturer's instructions. Specifically, the contents of a PowerBiofilm bead tube, and 350 μl of buffer BF1 and 100 μl of buffer BF2 were added to each sample tube. Samples were vortexed and incubated at 65°C for 5 min. Physical lysis of cell material was accomplished using the Mini-BeadBeater-16 (BioSpec Products, Bartlesville, OK) at 3450 oscillations/min for 2 min. Samples were spun at 13000 × g for 1 min. Supernatants were transferred to fresh tubes and 200 μl of buffer BF3 were added; samples were incubated at 4°C for 5 min and subsequently spun for 1 min. Supernatants were transferred to fresh tubes and 900 μl of buffer BF4 were added and samples mixed. Samples were loaded onto a PowerBiofilm spin filter column and spun for 1 min repeatedly until all sample was collected onto the filter. Filters were washed with 650 μl of buffer BF5 followed by buffer BF6 and ended with a final spin for 2 min. DNA was eluted in 100 μl of buffer BF7 with a final spin for 1 min.
Fujifilm QuickGene-Mini80 extraction platform
DNA was extracted from ten replicate subsamples of A, B, and C using the QuickGene DNA Tissue Kit S with the semi-automated QuickGene-Mini80 instrument (Autogen/FujiFilm, Holliston, MA) following manufacturer's instructions. Cell lysis was facilitated by adding 180 μl of Tissue lysis buffer (Autogen/FujiFilm) and 20 μl Proteinase K to each sample tube and mixing with a Thermolyne LabQuake Rotisserie Tube Shaker (ThermoScientific/Barnstead, Waltham, MA) for 30 min at 55°C. Samples were spun at 10000 × g for 3 min. The supernatants were transferred to a new tube and 20 μl RNase A were added and incubated for 2 min. Next, 180 μl Lysis buffer and 240 μl ethanol (>99%) were added and the sample was vortexed for 15 s. Samples were transferred to QuickGene cartridges and placed within the QuickGene Mini80 apparatus, and DNA binding, washing, and elution were accomplished through pressurization. DNA was eluted with 200 μl Elution buffer.
Promega Maxwell 16 extraction platform
DNA was extracted from ten replicate subsamples of A, B, and C using the automated Maxwell 16 Cell Total RNA Purification Kit with the Maxwell 16 Instrument (Promega) set to the LEV (low elution volume) configuration. Specifically, samples were loaded into the pre-dispensed reagent cartridges along with 400 μl RNA lysis buffer and 400 μl RNA dilution buffer. Elution tubes containing 100 μl nuclease-free water, plungers, and cartridges containing the sample and buffers were placed within the instrument and all subsequent steps were automated following the pre-programmed DNA extraction protocol. The DNA-removal steps of the Total RNA Purification Kit protocol were omitted to preserve the DNA (Promega Field Application Specialist, personal communication).
Evaluation of extracted DNA yield
DNA extracts from the subsamples were analyzed by gel electrophoresis and quantified using fluorometry to compare the reproducibility of extraction among replicate samples. To visualize the DNA fragment, 10 μl of each extract was analyzed alongside 2 μl of Lambda DNA/EcoRI+HindIII marker (ThermoFisherScientific/Fermentas, GlenBurnie, MD) on a 1% agarose gel (wt/vol) stained with SYBR®Safe (Invitrogen, Carlsbad, CA). Gels were visualized and the image captured using the Gel Logic 112 Imaging System and Molecular Imaging Software v5 (Carestream, WoodBridge, CT). DNA extracts were quantified using the Qubit 2.0 fluorometer with the dsDNA or RNA reagents according to the manufacturer’s protocol (Invitrogen/Life Technologies, Carlsbad, CA). GraphPad Prism5 (GraphPad Software, San Diego, CA) was used to generate box-and-whisker plots to visualize the degree of variation in DNA yields among replicate extractions. Upper and lower whiskers illustrate the maximum and minimum DNA yields, respectively, and the median DNA yield separates the box into upper (75%) and lower (25%) quartiles.
For each of the three platforms, the ten subsample DNA extracts were pooled to generate DNA stocks for subsequent analyses to assess DNA extract quality and its effect on the microbial community structure, while minimizing the effect of small sample volumes. For each of the three samples A, B, and C, the total amount of DNA extracted from equivalent sample volumes for each platform was determined by multiplying the concentration of each of the ten subsample extractions by its elution volume and summing the products.
Evaluation of extracted DNA quality using qPCR analysis
DNA extraction quality was evaluated for PCR inhibition via amplification of the bacterial 16S rRNA gene in undiluted versus diluted DNA extracts. Briefly, 30 μl reactions contained 15 μl 2 × SYBR®Green PCR Master Mix (Life Technologies, Carlsbad, CA), 0.5 M betaine (N,N,N-Trimethylglycine) (Sigma-Aldrich, St Louis, MO), 250 nM of the primer 27f (5′-AGAGTTTGATCCTGGCTCAG) and 125 nM of the primer 338r (5′- TGCTGCCTCCCGTAGGAGT) as described in Stevenson et al. (
). Thermal cycling, data acquisition and analyses were carried out with the StepOnePlus™ Real-Time PCR System and StepOne Software v2.1 (Life Technologies). Cycling conditions were: 95°C for 10 min followed by 40 cycles of 95°C for 30 s, 55°C for 45 s, 72°C for 45 s, and ended with a melt curve stage of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Image capture was at 72°C. DNA was assayed in triplicate at undiluted, 1:10, and 1:100 dilutions. A 10-fold dilution series of a control plasmid was assayed in duplicate and spanned 103
copies. Molar concentrations were converted into 16S copies based on the following assumptions: the average molecular mass of a double strand DNA base pair (bp) is 6.6 × 1011
, Avogadro’s number of copies mol-1
is 6.02 × 1023
(McKew and Smith
Evaluation of the influence of DNA extraction platform on microbial community composition using denaturing gradient gel electrophoresis (DGGE)
A DGGE analysis of amplified bacterial 16S rRNA genes was used to evaluate potential biases in cell lysis between extraction platforms. Briefly, 2 μl of DNA were amplified by end-point PCR in 25 μl reactions. Each reaction contained: 0.625 U DreamTaq™ polymerase (Fermentas, Glen Burnie, MD), 0.2 mM dNTP mixture, 0.5 M betaine, 1 × DreamTaq Buffer (Fermentas), and 100 nM each of forward primer GM5F (5′-CCTACGGGAGGCAGCAG) containing the GC clamp on the 5'-end and reverse primer D907R (5′-CCCCGTCAATTCCTTTGAGTTT) (Santegoeds et al.
1999). Thermal cycling was carried out with a TC-512 thermal cycler (Techne, Burlington, NJ) using a touchdown PCR program from 65°C to 55°C. Conditions were 94°C for 4 min, followed by 2 cycles each of 94°C for 1 min, N°C for 1 min, and 72°C for 1 min, where N°C dropped 1°C from 65°C to 55°C, followed by 15 cycles at the 55°C annealing temperature and a final extension at 72°C for 10 min. For bacterial community analysis, 15 μl of each reaction was resolved on a 6% polyacrylamide, urea and formamide 40-60% denaturing gradient gel (100% denaturant = 7 M Urea and 40% formamide) (Muyzer et al.
1993) and run at 65 V for 16 h at 60°C. The gel was stained for 15 min in SYBR Safe (at 25 μl per 250 ml) and visualized as described above.
Identification of microbial community composition among DNA extracts using high throughput sequencing of 16S rRNA gene libraries
To identify major bacterial taxa in samples A, B, and C DNA extracts, bacterial 16S rRNA gene libraries were generated from each extraction method modeled after the approach used by Wawrik et al. (
2011). For each sample, triplicate 50 μl reactions contained 5 μl to 10 μl DNA, 1.25 U DreamTaq polymerase, 0.2 mM dNTP mixture, 0.5 M betaine, 1xDreamTaq Buffer (Fermentas), 250 nM 27f and 125nM 338r primers. Thermal cycling was carried out on a TC-512 thermal cycler (Techne) with the following conditions: 96°C for 3 min; 30 cycles of 96°C for 30 s, 55°C for 45 s, 72°C for 45 s; and a final extension at 72°C for 10 min. Triplicate reactions were pooled and purified using the Wizard PCR Preps DNA Purification System (Promega). From each of the purified PCRs, 5 μl was added to a second PCR containing barcoded PCR primers TiA-8nt-CA-27f (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGxxxxxxxxCAAGAGTTTGATCCTGG CTCAG) and TiB-CA-338r (5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCA TGCTGCCTCCCGTAGGAGT) for multiplexed pyrosequencing as described by Hamady et al. (
2008). Each sample received a different tagged forward primer, containing a specific 8 nt ‘barcode’ sequence (designated by x), and samples were ‘tagged’ by re-amplification for six cycles. Barcodes are listed in Additional file
1: Table S1. The efficacy of the tagging reaction was confirmed by gel electrophoresis. Tagged PCR products were pooled in equimolar amounts and sequenced on a GS-FLX sequencer using the Titanium chemistry at (University of Oklahoma's Advanced Center for Genome Technology
The bacterial 16S rRNA gene libraries were analyzed using the bioinformatics software package, mothur ver1.24 (Schloss et al.
2009). An implementation of the Amplicon Noise algorithm was used to reduce the sequencing error incurred with pyrosequencing (Quince et al.
2011). Sequences were binned by barcode and screened to remove those containing errors in the forward primer or barcode. Unique sequences were trimmed to overlap a minimum of 200 base pairs and aligned against the SILVA reference alignment database (Pruesse et al.
2007) using the NAST-aligner (DeSantis et al.
2006). Sequences were pre-clustered using a single linkage algorithm (Huse et al.
2010) to reduce the number of spurious operational taxonomic units (OTUs) that would result from pyrosequencing errors, and subsequently screened for chimeras using UChime (Edgar et al.
2011). A distance matrix was generated and used to cluster sequences into OTUs at a 97% similarity level using the furthest neighbor algorithm. A representative sequence from each OTU was assigned a taxonomic classification based on the Ribosomal Database Project's naïve Bayesian classifier (Wang et al.
2007) at an 80% confidence threshold; and all richness and diversity measurements were calculated using the mothur software package based on a random subsampling subset of 1958 sequences to equal the number of reads in the smallest library. Using the generated distance matrix, an analysis of molecular variance (AMOVA) was used to determine if the observed differences in microbial diversity between sample groups or extraction methods was significantly different (Schloss
2008; Schloss and Handelsman
2008). Sequences were deposited in the short read archive of GenBank [GenBank: SRA052225].