Sequencing at sea: Challenges and experiences in Ion Torrent PGM sequencing during the 2013 Southern Line Islands Research Expedition
- Published
- Accepted
- Subject Areas
- Bioinformatics, Ecology, Genomics, Marine Biology, Microbiology
- Keywords
- genomics, sequencing, expeditions, metagenomics, environmental microbiology, coral reef, vibrio
- Copyright
- © 2014 Lim et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 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.433v1
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.
Author Comment
This paper is in review at PeerJ.
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.
