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Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

Oldham AL, Drilling HS, Stamps BW, Stevenson BS, Duncan KE - AMB Express (2012)

Bottom Line: The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition.Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach.Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Microbiology and Plant Biology, University of Oklahoma, 770 Van Vleet Oval GLCH #136, Norman, OK 73019, USA. athenia.L.oldham-1@ou.edu.

ABSTRACT
The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

No MeSH data available.


Variation among sample A replicate libraries from PowerBiofilm and Maxwell extractions. Relative abundance of bacterial phyla (class for Proteobacteria) from DNA extracted from sample A replicate libraries (n = 3 PowerBiofilm replicate libraries and n = 3 Maxwell replicate libraries). Unclassified sequences and phyla with membership < 1% of total sequences were pooled into the classification labeled "Other". AMOVA: p = 0.106.
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Figure 5: Variation among sample A replicate libraries from PowerBiofilm and Maxwell extractions. Relative abundance of bacterial phyla (class for Proteobacteria) from DNA extracted from sample A replicate libraries (n = 3 PowerBiofilm replicate libraries and n = 3 Maxwell replicate libraries). Unclassified sequences and phyla with membership < 1% of total sequences were pooled into the classification labeled "Other". AMOVA: p = 0.106.

Mentions: Next, to rule out extraction bias as the sole source of variation observed between extraction platforms, technical replicates were compared (Figure 5, Additional file 1: Table S2). Sample A was chosen for this analysis, as a large proportion of its membership belongs to the gram-positive Firmicutes (Figure 4a). Three replicates of the Maxwell and PowerBiofilm extractions were compared, as they represent the most and least automated platforms, respectively (Table 1). The six sample A libraries (three Maxwell replicates and three PowerBiofilm replicates) contained a total of 14806 sequences that clustered into 308 OTUs at 97% similarity, 127 of which were singletons (i.e. OTU containing a single sequence). Analysis of a random number of sequences (n = 1958) from each library showed that although there was variation among all libraries (Additional file 1 Table S2) they were not significantly different from one another (AMOVA: p = 0.106). Furthermore, an analysis of the dominant phyla demonstrated that sequences from all replicates were classified as members of the same few genera: Thermacetogenium, Halolactibacillus, and Thermoanaerobacter for Firmicutes (phylum); Thermovirga for Synergistetes (phylum); Thermodesulfobacterium for Thermodesulfobacteria (phylum); and Kosmotoga, Thermotoga, and Thermosipho for Thermotogae (phylum). Approximately 99% of the sequences were represented in both extraction methods. The ~1% of sequences exclusive to one or the other were either unclassified or sequences present in only one of the three replicates for a given extraction method.


Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

Oldham AL, Drilling HS, Stamps BW, Stevenson BS, Duncan KE - AMB Express (2012)

Variation among sample A replicate libraries from PowerBiofilm and Maxwell extractions. Relative abundance of bacterial phyla (class for Proteobacteria) from DNA extracted from sample A replicate libraries (n = 3 PowerBiofilm replicate libraries and n = 3 Maxwell replicate libraries). Unclassified sequences and phyla with membership < 1% of total sequences were pooled into the classification labeled "Other". AMOVA: p = 0.106.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539857&req=5

Figure 5: Variation among sample A replicate libraries from PowerBiofilm and Maxwell extractions. Relative abundance of bacterial phyla (class for Proteobacteria) from DNA extracted from sample A replicate libraries (n = 3 PowerBiofilm replicate libraries and n = 3 Maxwell replicate libraries). Unclassified sequences and phyla with membership < 1% of total sequences were pooled into the classification labeled "Other". AMOVA: p = 0.106.
Mentions: Next, to rule out extraction bias as the sole source of variation observed between extraction platforms, technical replicates were compared (Figure 5, Additional file 1: Table S2). Sample A was chosen for this analysis, as a large proportion of its membership belongs to the gram-positive Firmicutes (Figure 4a). Three replicates of the Maxwell and PowerBiofilm extractions were compared, as they represent the most and least automated platforms, respectively (Table 1). The six sample A libraries (three Maxwell replicates and three PowerBiofilm replicates) contained a total of 14806 sequences that clustered into 308 OTUs at 97% similarity, 127 of which were singletons (i.e. OTU containing a single sequence). Analysis of a random number of sequences (n = 1958) from each library showed that although there was variation among all libraries (Additional file 1 Table S2) they were not significantly different from one another (AMOVA: p = 0.106). Furthermore, an analysis of the dominant phyla demonstrated that sequences from all replicates were classified as members of the same few genera: Thermacetogenium, Halolactibacillus, and Thermoanaerobacter for Firmicutes (phylum); Thermovirga for Synergistetes (phylum); Thermodesulfobacterium for Thermodesulfobacteria (phylum); and Kosmotoga, Thermotoga, and Thermosipho for Thermotogae (phylum). Approximately 99% of the sequences were represented in both extraction methods. The ~1% of sequences exclusive to one or the other were either unclassified or sequences present in only one of the three replicates for a given extraction method.

Bottom Line: The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition.Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach.Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Microbiology and Plant Biology, University of Oklahoma, 770 Van Vleet Oval GLCH #136, Norman, OK 73019, USA. athenia.L.oldham-1@ou.edu.

ABSTRACT
The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

No MeSH data available.