Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Sanne Boessenkool; Laura S. Epp; James Seymour Haile; Eva Bellemain; Mary Edwards; Eric Coissac; Eske Willerslev; Christian Brochmann

doi:10.1111/j.1365-294X.2011.05306.x

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Sanne Boessenkool, Laura S. Epp, James Seymour Haile, Eva Bellemain, Mary Edwards, Eric Coissac, Eske Willerslev, Christian Brochmann

68 Citationer (Scopus)

Abstract

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 because of 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 optimized 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 (Coelodonta 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 analyses, the PCR can be biased towards the 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.

Originalsprog	Engelsk
Tidsskrift	Molecular Ecology
Vol/bind	21
Udgave nummer	8
Sider (fra-til)	1806-1815
Antal sider	10
ISSN	0962-1083
DOI	https://doi.org/10.1111/j.1365-294X.2011.05306.x
Status	Udgivet - apr. 2012

Adgang til dokumentet

10.1111/j.1365-294X.2011.05306.x

Citationsformater

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title = "Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA",

abstract = "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 because of 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 optimized 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 (Coelodonta 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 analyses, the PCR can be biased towards the 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.",

author = "Sanne Boessenkool and Epp, {Laura S.} and Haile, {James Seymour} and Eva Bellemain and Mary Edwards and Eric Coissac and Eske Willerslev and Christian Brochmann",

note = "Special Issue: Environmental DNA",

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T1 - Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

AU - Boessenkool, Sanne

AU - Epp, Laura S.

AU - Haile, James Seymour

AU - Bellemain, Eva

AU - Edwards, Mary

AU - Coissac, Eric

AU - Willerslev, Eske

AU - Brochmann, Christian

N1 - Special Issue: Environmental DNA

PY - 2012/4

Y1 - 2012/4

N2 - 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 because of 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 optimized 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 (Coelodonta 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 analyses, the PCR can be biased towards the 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.

AB - 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 because of 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 optimized 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 (Coelodonta 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 analyses, the PCR can be biased towards the 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.

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DO - 10.1111/j.1365-294X.2011.05306.x

M3 - Journal article

C2 - 21988749

SN - 0962-1083

VL - 21

SP - 1806

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JO - Molecular Ecology

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