Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples
A degenerate PCR-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. The method, which we have called Random Amplification Metagenomic PCR (RAMP), involves the use of specific pr...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2012-01-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2012.00017/full |
| _version_ | 1811307086915567616 |
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| author | Amanda eMartino Matthew E. Rhodes Jennifer F Biddle Leah D. Brandt Lynn P. Tomsho Christopher H House |
| author_facet | Amanda eMartino Matthew E. Rhodes Jennifer F Biddle Leah D. Brandt Lynn P. Tomsho Christopher H House |
| author_sort | Amanda eMartino |
| collection | DOAJ |
| description | A degenerate PCR-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. The method, which we have called Random Amplification Metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3’ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10X. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa show that community analysis can be sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low biomass samples. |
| first_indexed | 2024-04-13T08:58:22Z |
| format | Article |
| id | doaj.art-03ceda0cf6b34a65b7af3d77e5344823 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-13T08:58:22Z |
| publishDate | 2012-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-03ceda0cf6b34a65b7af3d77e53448232022-12-22T02:53:13ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2012-01-01310.3389/fmicb.2012.0001717378Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samplesAmanda eMartino0Matthew E. Rhodes1Jennifer F Biddle2Leah D. Brandt3Lynn P. Tomsho4Christopher H House5Pennsylvania State UniversityPennsylvania State UniversityUniversity of Delaware College of Earth Ocean and EnvironmentPennsylvania State UniversityPennsylvania State UniversityPennsylvania State UniversityA degenerate PCR-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. The method, which we have called Random Amplification Metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3’ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10X. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa show that community analysis can be sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low biomass samples.http://journal.frontiersin.org/Journal/10.3389/fmicb.2012.00017/fullArchaeaBacteriaMetagenomicsdeep biosphereocean drillinglow-biomass |
| spellingShingle | Amanda eMartino Matthew E. Rhodes Jennifer F Biddle Leah D. Brandt Lynn P. Tomsho Christopher H House Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples Frontiers in Microbiology Archaea Bacteria Metagenomics deep biosphere ocean drilling low-biomass |
| title | Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples |
| title_full | Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples |
| title_fullStr | Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples |
| title_full_unstemmed | Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples |
| title_short | Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples |
| title_sort | novel degenerate pcr method for whole genome amplification applied to peru margin odp leg 201 subsurface samples |
| topic | Archaea Bacteria Metagenomics deep biosphere ocean drilling low-biomass |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2012.00017/full |
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