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Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor

Ussler, William, III and Preston, Christina and Tavormina, Patricia and Pargett, Doug and Jensen, Scott and Roman, Brent and Marin, Roman, III and Shah, Sunita R. and Girguis, Peter R. and Birch, James M. and Orphan, Victoria and Scholin, Christopher (2013) Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor. Environmental Science and Technology, 47 (16). pp. 9339-9346. ISSN 0013-936X. doi:10.1021/es4023199.

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Recent advances in ocean observing systems and genomic technologies have led to the development of the deep-sea environmental sample processor (D-ESP). The DESP filters particulates from seawater at depths up to 4000 m and applies a variety of molecular assays to the particulates, including quantitative PCR (qPCR), to identify particular organisms and genes in situ. Preserved samples enable laboratory-based validation of in situ results and expanded studies of genomic diversity and gene expression. Tests of the D-ESP at a methane-rich mound in the Santa Monica Basin centered on detection of 16S rRNA and particulate methane monooxygenase (pmoA) genes for two putative aerobic methanotrophs. Comparison of in situ qPCR results with laboratory-based assays of preserved samples demonstrates the D-ESP generated high-quality qPCR data while operating autonomously on the seafloor. Levels of 16S rRNA and pmoA cDNA detected in preserved samples are consistent with an active community of aerobic methanotrophs near the methane-rich mound. These findings are substantiated at low methane sites off Point Conception and in Monterey Bay where target genes are at or below detection limits. Successful deployment of the D-ESP is a major step toward developing autonomous systems to facilitate a wide range of marine microbiological investigations.

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Orphan, Victoria0000-0002-5374-6178
Additional Information:Copyright © 2013 American Chemical Society. Received: May 24, 2013; Revised: July 24, 2013; Accepted: July 24, 2013; Published: July 24, 2013. Development and application of ESP technology has been funded in part by grants from the David and Lucile Packard Foundation through funds allocated by the Monterey Bay Aquarium Research Institute (MBARI), NSF (OCE-0314222 and EF-0424599), NASA Astrobiology (NNG06GB34G, NNX09AB78G), Keck Foundation (by subcontract from University of Washington, Seattle), and Gordon and Betty Moore Foundation (ERG731). Elif Demir-Hilton, Kevan Yamahara, and three anonymous reviewers provided constructive comments and feedback on earlier versions of this manuscript. We thank the engineering technicians and machinists at MBARI for their invaluable help and dedication toward instrument development and the crews and ROV pilots of the R/Vs Point Lobos and Western Flyer for their support and expertise during field operations. The authors declare no competing financial interest. Supporting Information: Detailed descriptions of qPCR assay development, nucleic acid extraction, ISMS operation and calibration, and dissolved methane concentration analysis. Figure S1-S2 shows characteristic features of the seafloor at the MARS deployment site. Figure S2-S11 illustrates water column methane concentration profiles from above the Santa Monica mound. Table S1-S5 lists primer and probe sequences used for the qPCR 5′-nuclease assays. Table S2-S8 summarizes qPCR standard curve parameters for in situ and postdeployment gene expression assays. This material is available free of charge via the Internet at
Funding AgencyGrant Number
David and Lucile Packard FoundationUNSPECIFIED
Monterey Bay Aquarium Research InstituteUNSPECIFIED
W. M. Keck FoundationUNSPECIFIED
Gordon and Betty Moore FoundationERG731
Issue or Number:16
Record Number:CaltechAUTHORS:20140723-162327897
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Official Citation:Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor William Ussler, III, Christina Preston, Patricia Tavormina, Doug Pargett, Scott Jensen, Brent Roman, Roman Marin, III, Sunita R. Shah, Peter R. Girguis, James M. Birch, Victoria Orphan, and Christopher Scholin Environmental Science & Technology 2013 47 (16), 9339-9346
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:47452
Deposited By: George Porter
Deposited On:24 Jul 2014 15:14
Last Modified:10 Nov 2021 17:39

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