Preparation and Analysis of Metatranscriptomic Libraries in Petroleum Hydrocarbon Microbe Systems

Hampton-Marcell, Jarrad; Frazier, Angel; Moormann, Stephanie M.; Owens, Sarah M.; Gilbert, Jack A.

doi:10.1007/8623_2014_24

Cited by 2 publications

(3 citation statements)

References 14 publications

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“…The main criteria for this choice were (i) compatibility with the DNA extraction protocol, (ii) the effective DNA stabilization capacity over a range of temperatures and (iii) no restrictive chemicals in relation to shipping. As a number of studies have previously shown no substantial differences in DNA preservation efficacy between the two buffers (Gomez‐Silvan et al, 2018) and they are referred to as equivalent in standardized genetic and genomic protocols (Duran & Cravo‐Laureau, 2017; Hampton‐Marcell et al, 2017), we allowed sample providers to select either. The somewhat higher variance in metabarcoding data from the LifeGuard samples was largely determined by the presence of a few outliers.…”

Section: Discussionmentioning

confidence: 99%

Towards reproducible metabarcoding data: Lessons from an international cross‐laboratory experiment

Zaiko

Greenfield

Abbott

et al. 2021

Molecular Ecology Resources

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Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (

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Section: Discussionmentioning

confidence: 99%

Towards reproducible metabarcoding data: Lessons from an international cross‐laboratory experiment

Zaiko

Greenfield

Abbott

et al. 2021

Molecular Ecology Resources

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“…Metatranscriptomic analysis has been evaluated with data from three studies. This analysis is considered a powerful technique that enables the examination of the gene expression profile of microorganisms present in a sample, providing valuable insights into their metabolic activity in a given environment [64]. Despite the advancements in microbiome studies of oilfield samples, there is still a notable scarcity of studies analyzing the metatranscriptome, and this can be attributed to several challenges, including the extraction of high-quality RNA, the presence of inhibitors, limited quantities of genetic material in the samples, and rapid RNA degradation.…”

Section: Rna Amplification and Sequencing Methodsmentioning

confidence: 99%

“…RNA transcript sequencing is limited by difficulties in preserving and isolating highquality RNA from environmental samples as RNA is an extremely unstable molecule and susceptible to degradation by the action of RNase enzymes [22]. Moreover, an adequate amount of RNA is critical to successfully carry out all stages of the analytical process, which involves the synthesis of complementary DNA (cDNA), construction of cDNA libraries, sequencing, and bioinformatic processing of obtained sequences [29]. Consequently, there is a lack of studies reporting the microbial diversity and composition in oil industry samples based on RNA transcripts [22].…”

Section: Introductionmentioning

confidence: 99%

Metabolically Active Microbial Communities in Oilfields: A Systematic Review and Synthesis of RNA Preservation, Extraction, and Sequencing Methods

Gomes,

García,

Dutra

et al. 2023

Applied Microbiology

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Characterizing metabolically active microorganisms using RNA-based methods is a crucial tool for monitoring and mitigating operational issues, such as oil biodegradation and biocorrosion of pipelines in the oil and gas industry. Our review, a pioneering study, addresses the main methods used to preserve, isolate, and sequence RNA from oilfield samples and describes the most abundant metabolically active genera studied. Using the MEDLINE/PubMed, PubMed Central, Scopus, and Web of Science databases, 2.561 potentially eligible records were identified. After screening, 20 studies were included in our review, underscoring the scarcity of studies related to the subject. Data were extracted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). These studies evaluated different samples, including produced water (PW), injection water (IW), solid deposits (SD), oil (OIL), and oily sludge (OS) collected from oilfields located in Australia, China, India, Mexico, and the United Arab Emirates. Environmental samples accounted for 55% of the studies, while enriched cultures and microbial consortia represented 35% and 15% of studies, respectively. PW was the most frequently studied sample, comprising 72% of all samples. Filtration and centrifugation were the only processes employed to concentrate the biomass present in samples. For RNA preservation, the most used method was a solution composed of 95:5 v/v ethanol/TRIzol, while for RNA isolation, the TRIzol reagent was the most cited. The Sanger sequencing method was used in all studies evaluating functional genes (alkB, dsrA, aprA, assA, and mcrA), and the Next-Generation Sequencing (NGS) method was employed in studies for sequencing transcripts of the 16S rRNA gene and metatranscriptomes. Pseudomonas (16S rRNA = PW: 2%; IW: 8%; metatranscriptome = PW: 20%) and Acinetobacter (16S rRNA = PW: 1%; IW: 4%; metatranscriptome = PW: 17%) were the most abundant genera. This study outlined the primary methods employed in researching metabolically active microorganisms. These data provide a foundation for future research. However, it is essential to note that we cannot yet determine the most effective method. We hope that this study will inspire further research related to the standardization of RNA preservation, extraction, and sequencing methods and significantly contribute to our understanding of active microbial communities in oilfields.

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Preparation and Analysis of Metatranscriptomic Libraries in Petroleum Hydrocarbon Microbe Systems

Cited by 2 publications

References 14 publications

Towards reproducible metabarcoding data: Lessons from an international cross‐laboratory experiment

Towards reproducible metabarcoding data: Lessons from an international cross‐laboratory experiment

Metabolically Active Microbial Communities in Oilfields: A Systematic Review and Synthesis of RNA Preservation, Extraction, and Sequencing Methods

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