Sequencing at sea: Challenges and experiences in Ion Torrent PGM sequencing during the 2013 Southern Line Islands Research Expedition
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Abstract
Genomics and metagenomics have revolutionized our understanding of marine microbial ecology and the importance of microbes in global geochemical cycles. However, the process of DNA sequencing has always been an abstract extension of the research expedition, completed once the samples were returned to the laboratory. During the 2013 Southern Line Islands Research Expedition, we started the first effort to bring next generation sequencing to some of the most remote locations on our planet. We successfully sequenced twenty six marine microbial genomes, and two marine microbial metagenomes using the Ion Torrent PGM platform on the Merchant Yacht Hanse Explorer. Onboard sequence assembly, annotation, and analysis enabled us to investigate the role of the microbes in the coral reef ecology of these islands and atolls. This analysis identified phospohonate as an important phosphorous source for microbes growing in the Line Islands and reinforced the importance of L-serine in marine microbial ecosystems. Sequencing in the field allowed us to propose hypotheses and conduct experiments and further sampling based on the sequences generated. By eliminating the delay between sampling and sequencing, we enhanced the productivity of the research expedition. By overcoming the hurdles associated with sequencing on a boat in the middle of the Pacific Ocean we proved the flexibility of the sequencing, annotation, and analysis pipelines.
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2014. Sequencing at sea: Challenges and experiences in Ion Torrent PGM sequencing during the 2013 Southern Line Islands Research Expedition. PeerJ PrePrints 2:e433v1 https://doi.org/10.7287/peerj.preprints.433v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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Supplemental Information
Media formulations in phenotype assay plates
Supplemental Table 1: In-house phenotypic array plate. Each stock well contains 1ml of 6X basal media (6X MOPS media, 57 mM NH4Cl, 1.5 mM NaSO4, 30 μM CaCl2, 6 mM MgSO4, 1.9M NaCl, 7.92 mM K2HPO4, 60 mM KCl, 36 μM FeCl3) and 1ml of 5X carbon substrate. The substrates are used at a final concentration of 0.2% unless specified.
Orthologous groups in all the genomes
Supplemental Table 2. Orthologous groups identified in all the genomes. OG is a unique identifier; Function is the most common function annotated for all the proteins; The remaining columns identify the protein encoding genes (pegs) found in each genome or None if the gene is not present.
2013 Southern Line Islands Research Expedition
Supplemental Figure 1: Expedition route of the 2013 Southern Line Island Expedition. The expedition left Papeete Harbor and circumnavigated the Southern Line Islands in a clockwise direction.
Sampling the reefs
Supplemental Figure 2: Examples of sampling surface for bacterial isolates. VRT35 and VRT38 were isolated from coral surfaces, while VRT37 and VRT41 were isolated from algae surfaces.
Number of bases sequenced and number of contigs generated
Supplemental Figure 3. Number of bases sequenced and number of contigs generated. The best-fit power regression of the data (shown with a gray line) has the equation [number of contigs] = 3.87 * 1010 [base piars sequenced]-0.95 with an R2 of 0.667.
Growth curves in 48 different media showing growth in some wells
Supplemental Figure 4: Growth curve of bacterial isolates in 48 different carbon sources indicated in Supplementary Table 1.
Heat map showing growth characteristics of all the isolates
Supplemental Figure 5: Heatmap showing the growth characteristics of each bacterial isolates from the 2013 Southern Line Island expedition in 48 different carbon sources.
Negative controls don't grow
Supplemental Figure 6: Growth curve of negative controls and filtered seawater-only samples in 48 different carbon sources indicated in Supplementary Table 1.
There is no correlation between genetic and physical distance
Supplemental Fig 7. Correlation between physical distance and genetic distance in the microbial species purified on the Line Islands.
Additional Information
Competing Interests
Timothy Harkins, Clarence Lee, and Warren Tom are employees of Life Technologies, Inc. and that Mark JA Vermeij is an employee of the Caribbean Research and Management of Biodiversity (CARMABI).
Author Contributions
Yan Wei Lim conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Daniel A Cuevas analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Genivaldo Gueiros Z. Silva analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Kristen Aguinaldo analyzed the data, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Elizabeth Dinsdale contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Andreas F Haas performed the experiments, analyzed the data, reviewed drafts of the paper.
Mark Hatay contributed reagents/materials/analysis tools, prepared figures and/or tables, reviewed drafts of the paper.
Savannah E Sanchez contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Linda W. Kelly analyzed the data, reviewed drafts of the paper.
Bas E. Dutilh analyzed the data, reviewed drafts of the paper.
Timothy T. Harkins contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Clarence C Lee contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Warren Tom contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Stuart A Sandin reviewed drafts of the paper, organized the field expedition, permits, and logistics.
Jennifer Smith reviewed drafts of the paper, organized the field expedition, permits, and logistics.
Brian Zgliczynski reviewed drafts of the paper, organized the field expedition, permits, and logistics.
Mark JA Vermeij performed the experiments, reviewed drafts of the paper.
Forest Rohwer conceived and designed the experiments, performed the experiments, reviewed drafts of the paper.
Robert Edwards conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers):
All samples were collected on SCUBA and under the Scientific Research Permit 021/13 issued by the Environment and Conservation Division of the Republic of Kiribati.
Grant Disclosures
The following grant information was disclosed by the authors:
NSF DEB-1046413, NSF CNS-1305112, NSF MCB-1330800
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
All sequences are in NCBI Bioproject PRJNA253472
Individual sequences:
NCBI BioSample Database
SAMN02904893
SAMN02904892
SAMN02870694
SAMN02870695
SAMN02870696
SAMN02870697
SAMN02870670
SAMN02870717
SAMN02870718
SAMN02870712
SAMN02870698
SAMN02870713
SAMN02870714
SAMN02870715
SAMN02870699
SAMN02870716
SAMN02870706
SAMN02870707
SAMN02870710
SAMN02870700
SAMN02870711
SAMN02870701
SAMN02870702
SAMN02870703
SAMN02870704
SAMN02870708
SAMN02870705
SAMN02870709
Data Deposition
The following information was supplied regarding the deposition of related data:
We will deposit figures in FigShare
Funding
This work is partially supported by NSF Dimensions Grant (DEB-1046413; Edwards and Rohwer). This project was also funded in part by the Gordon and Betty Moore Foundation through Grant GBMF-3781 to Rohwer. Additional funding for Yanwei Lim was provided by the Canadian Institute for Advanced Research (CIFAR; IMB-ROHW-141679). Additional funding for Edwards was provided by NSF grants CNS-1305112, and MCB-1330800. Dutilh was supported by an award from CAPES/BRASIL. The SDSU Vice President of Research, Director's Office of Scripps Institution of Oceanography, Moore Family Foundation, and several private donors provided cruise support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.