Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA
Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results due to competition, or bias,...
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ftdryad:oai:v1.datadryad.org:10255/dryad.34990 2023-05-15T17:57:12+02:00 Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA Boessenkool, Sanne Epp, Laura S. Haile, James Bellemain, Eva Edwards, Mary Coissac, Eric Willerslev, Eske Brochmann, Christian 2011-09-09T15:51:51Z http://hdl.handle.net/10255/dryad.34990 https://doi.org/10.5061/dryad.t33v4k14 unknown doi:10.5061/dryad.t33v4k14/1 doi:10.5061/dryad.t33v4k14/2 doi:10.1111/j.1365-294X.2011.05306.x PMID:21988749 doi:10.5061/dryad.t33v4k14 Boessenkool S, Epp LS, Haile J, Bellemain E, Edwards M, Coissac E, Willerslev E, Brochmann C (2011) Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. Molecular Ecology 21(8): 1806–1815. http://hdl.handle.net/10255/dryad.34990 Bioinfomatics/Phyloinfomatics Mammals DNA Barcoding Genomics/Proteomics Article 2011 ftdryad https://doi.org/10.5061/dryad.t33v4k14 https://doi.org/10.5061/dryad.t33v4k14/1 https://doi.org/10.5061/dryad.t33v4k14/2 https://doi.org/10.1111/j.1365-294X.2011.05306.x 2020-01-01T14:54:17Z Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results due to competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA we first optimised the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (C. antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analysis, the PCR can be biased towards amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified. Article in Journal/Newspaper permafrost Siberia Dryad Digital Repository (Duke University) |
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Open Polar |
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Dryad Digital Repository (Duke University) |
op_collection_id |
ftdryad |
language |
unknown |
topic |
Bioinfomatics/Phyloinfomatics Mammals DNA Barcoding Genomics/Proteomics |
spellingShingle |
Bioinfomatics/Phyloinfomatics Mammals DNA Barcoding Genomics/Proteomics Boessenkool, Sanne Epp, Laura S. Haile, James Bellemain, Eva Edwards, Mary Coissac, Eric Willerslev, Eske Brochmann, Christian Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
topic_facet |
Bioinfomatics/Phyloinfomatics Mammals DNA Barcoding Genomics/Proteomics |
description |
Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results due to competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA we first optimised the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (C. antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analysis, the PCR can be biased towards amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified. |
format |
Article in Journal/Newspaper |
author |
Boessenkool, Sanne Epp, Laura S. Haile, James Bellemain, Eva Edwards, Mary Coissac, Eric Willerslev, Eske Brochmann, Christian |
author_facet |
Boessenkool, Sanne Epp, Laura S. Haile, James Bellemain, Eva Edwards, Mary Coissac, Eric Willerslev, Eske Brochmann, Christian |
author_sort |
Boessenkool, Sanne |
title |
Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_short |
Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_full |
Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_fullStr |
Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_full_unstemmed |
Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_sort |
data from: blocking human contaminant dna during pcr allows amplification of rare mammal species from sedimentary ancient dna |
publishDate |
2011 |
url |
http://hdl.handle.net/10255/dryad.34990 https://doi.org/10.5061/dryad.t33v4k14 |
genre |
permafrost Siberia |
genre_facet |
permafrost Siberia |
op_relation |
doi:10.5061/dryad.t33v4k14/1 doi:10.5061/dryad.t33v4k14/2 doi:10.1111/j.1365-294X.2011.05306.x PMID:21988749 doi:10.5061/dryad.t33v4k14 Boessenkool S, Epp LS, Haile J, Bellemain E, Edwards M, Coissac E, Willerslev E, Brochmann C (2011) Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. Molecular Ecology 21(8): 1806–1815. http://hdl.handle.net/10255/dryad.34990 |
op_doi |
https://doi.org/10.5061/dryad.t33v4k14 https://doi.org/10.5061/dryad.t33v4k14/1 https://doi.org/10.5061/dryad.t33v4k14/2 https://doi.org/10.1111/j.1365-294X.2011.05306.x |
_version_ |
1766165585760092160 |