CoChat
Sign Up
Cited by 56Forensic and Genetic ResearchGenomics and Phylogenetic StudiesGenetic diversity and population structure

Road blocks on paleogenomes—polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

Open access
Heyn, Patricia, Stenzel, Udo, Briggs, Adrian W., Kircher, Martin, Hofreiter, Michael, Meyer, Matthias·2010·Nucleic Acids Research
DOI 10.1093/nar/gkq572PMC PMC2938203
Open this paper in CoChat — free account

Abstract

Although the last few years have seen great progress in DNA sequence retrieval from fossil specimens, some of the characteristics of ancient DNA remain poorly understood. This is particularly true for blocking lesions, i.e. chemical alterations that cannot be bypassed by DNA polymerases and thus prevent amplification and subsequent sequencing of affected molecules. Some studies have concluded that the vast majority of ancient DNA molecules carry blocking lesions, suggesting that the removal, repair or bypass of blocking lesions might dramatically increase both the time depth and geographical range of specimens available for ancient DNA analysis. However, previous studies used very indirect detection methods that did not provide conclusive estimates on the frequency of blocking lesions in endogenous ancient DNA. We developed a new method, polymerase extension profiling (PEP), that directly reveals occurrences of polymerase stalling on DNA templates. By sequencing thousands of single primer extension products using PEP methodology, we have for the first time directly identified blocking lesions in ancient DNA on a single molecule level. Although we found clear evidence for blocking lesions in three out of four ancient samples, no more than 40% of the molecules were affected in any of the samples, indicating that such modifications are far less frequent in ancient DNA than previously thought.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1093/nar/gkq572

Cites

2 of 2 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Previous studies have reported lower error rates and higher extension termination rates for Pfu-based polymerases (such as Phusion) compared to Taq-based and other uracil-tolerant polymerases.

    “Previous studies have reported lower error rates and higher extension termination rates for Pfu‐based polymerases (such as Phusion) compared to Taq‐based and other uracil‐tolerant polymerases (Heyn et al., 2010; Ginolhac et al., 2011; Wales et al., 2014).”

    From Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus (2016) · cites this paper as Heyn et al., 2010

    In Results, ‘PEP on artificial templates’ (page not provided), Pfu showed much higher extension termination on uracil-containing templates than the Taq-based polymerases tested. The paper calls Pfu a proofreading enzyme, but does not report comparative error rates or test Phusion; its termination finding is also specific to the lesions and templates studied. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Platinum Taq HiFi has been shown to efficiently amplify over >90% of uracil sites.

    “Platinum Taq HiFi has also been shown to efficiently amplify over >90% of uracil sites (Heyn et al., 2010).”

    From Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus (2016) · cites this paper as Heyn et al., 2010

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 38

  1. [1]Miller W Drautz DI Ratan A Pusey B Qi J Lesk AM Tomsho LP Packard MD Zhao F Sher A Sequencing the nuclear genome of the extinct woolly mammoth Nature 2008 456 387 390 19020620
  2. [3]Green RE Krause J Briggs AW Maricic T Stenzel U Kircher M Patterson N Li H Zhai W Fritz MH A draft sequence of the Neandertal genome Science 2010 328 710 722 20448178 PMC5100745
  3. [4]Briggs AW Good JM Green RE Krause J Maricic T Stenzel U Lalueza-Fox C Rudan P Brajkovic D Kucan Z Targeted retrieval and analysis of five Neandertal mtDNA genomes Science 2009 325 318 321 19608918
  4. [5]Stiller M Knapp M Stenzel U Hofreiter M Meyer M Direct multiplex sequencing (DMPS)—a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA Genome Res. 2009 19 1843 1848 19635845 PMC2765274
  5. [6]Burbano HA Hodges E Green RE Briggs AW Krause J Meyer M Good JM Maricic T Johnson PL Xuan Z Targeted investigation of the Neandertal genome by array-based sequence capture Science 2010 328 723 725 20448179 PMC3140021
  6. [7]Margulies M Egholm M Altman WE Attiya S Bader JS Bemben LA Berka J Braverman MS Chen YJ Chen Z Genome sequencing in microfabricated high-density picolitre reactors Nature 2005 437 376 380 16056220 PMC1464427
  7. [8]Bentley DR Balasubramanian S Swerdlow HP Smith GP Milton J Brown CG Hall KP Evers DJ Barnes CL Bignell HR Accurate whole human genome sequencing using reversible terminator chemistry Nature 2008 456 53 59 18987734 PMC2581791
  8. [9]Briggs AW Stenzel U Johnson PL Green RE Kelso J Prüfer K Meyer M Krause J Ronan MT Lachmann M Patterns of damage in genomic DNA sequences from a Neandertal Proc. Natl Acad. Sci. USA 2007 104 14616 14621 17715061 PMC1976210
  9. [10]Brotherton P Endicott P Sanchez JJ Beaumont M Barnett R Austin J Cooper A Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of postmortem miscoding lesions Nucleic Acids Res. 2007 35 5717 5728 17715147 PMC2034480
  10. [11]Briggs AW Stenzel U Meyer M Krause J Kircher M Pääbo S Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA Nucleic Acids Res. 2010 38 e87 20028723 PMC2847228
  11. [12]Pääbo S Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification Proc. Natl Acad. Sci. USA 1989 86 1939 1943 2928314 PMC286820
  12. [13]Höss M Jaruga P Zastawny TH Dizdaroglu M Pääbo S DNA damage and DNA sequence retrieval from ancient tissues Nucleic Acids Res. 1996 24 1304 1307 8614634 PMC145783
  13. [14]Hansen AJ Mitchell DL Wiuf C Paniker L Brand TB Binladen J Gilichinsky DA Ronn R Willerslev E Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments Genetics 2006 173 1175 1179 16582426 PMC1526502
  14. [15]d'Abbadie M Hofreiter M Vaisman A Loakes D Gasparutto D Cadet J Woodgate R Pääbo S Holliger P Molecular breeding of polymerases for amplification of ancient DNA Nat. Biotechnol. 2007 25 939 943 17632524 PMC1978225
  15. [16]Gloeckner C Sauter KB Marx A Evolving a thermostable DNA polymerase that amplifies from highly damaged templates Angew. Chem. Int. Ed. Engl. 2007 46 3115 3117 17366498
  16. [17]Binladen J Gilbert MT Bollback JP Panitz F Bendixen C Nielsen R Willerslev E The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing PLoS ONE 2007 2 e197 17299583 PMC1797623
  17. [18]Rohland N Hofreiter M Ancient DNA extraction from bones and teeth Nat. Protoc. 2007 2 1756 1762 17641642
  18. [19]DeAngelis MM Wang DG Hawkins TL Solid-phase reversible immobilization for the isolation of PCR products Nucleic Acids Res. 1995 23 4742 4743 8524672 PMC307455
  19. [20]Blondal T Hjorleifsdottir SH Fridjonsson OF Aevarsson A Skirnisdottir S Hermannsdottir AG Hreggvidsson GO Smith AV Kristjansson JK Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1 Nucleic Acids Res. 2003 31 7247 7254 14654700 PMC291858
  20. [21]Li TW Weeks KM Structure-independent and quantitative ligation of single-stranded DNA Anal. Biochem. 2006 349 242 246 16325753
  21. [22]Meyer M Briggs AW Maricic T Höber B Höffner B Krause J Weihmann A Pääbo S Hofreiter M From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing Nucleic Acids Res. 2008 36 e5 18084031 PMC2248761
  22. [23]Green RE Malaspinas AS Krause J Briggs AW Johnson PL Uhler C Meyer M Good JM Maricic T Stenzel U A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing Cell 2008 134 416 426 18692465 PMC2602844
  23. [24]Lawyer FC Stoffel S Saiki RK Myambo K Drummond R Gelfand DH Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus J. Biol. Chem. 1989 264 6427 6437 2649500
  24. [25]Lundberg KS Shoemaker DD Adams MW Short JM Sorge JA Mathur EJ High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus Gene 1991 108 1 6 1761218
  25. [26]Boudsocq F Iwai S Hanaoka F Woodgate R Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poleta Nucleic Acids Res. 2001 29 4607 4616 11713310 PMC92520
  26. [27]Lu YY Ye SY Hong GF Large fragment of DNA polymerase I from Bacillus stearothermophilus (Bst polymerase) is stable at ambient temperature Biotechniques 1991 11 464, 466 1793578
  27. [28]Olsen DB Eckstein F Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase: exploitation for DNA sequencing Nucleic Acids Res 1989 17 9613 9620 2602138 PMC335201
  28. [29]Lindahl T Andersson A Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid Biochemistry 1972 11 3618 3623 4559796
  29. [30]Wellinger RE Thoma F Taq DNA polymerase blockage at pyrimidine dimers Nucleic Acids Res. 1996 24 1578 1579 8628696 PMC145810
  30. [31]Pfeifer GP Formation and processing of UV photoproducts: effects of DNA sequence and chromatin environment Photochem. Photobiol. 1997 65 270 283 9066304
  31. [33]Krause J Briggs AW Kircher M Maricic T Zwyns N Derevianko A Pääbo S A complete mtDNA genome of an early modern human from Kostenki, Russia Curr. Biol. 2010 20 231 236 20045327
  32. [34]Greagg MA Fogg MJ Panayotou G Evans SJ Connolly BA Pearl LH A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil Proc. Natl Acad. Sci. USA 1999 96 9045 9050 10430892 PMC17729
  33. [35]Strauss BS The “A” rule revisited: polymerases as determinants of mutational specificity DNA Repair (Amsterdam) 2002 1 125 135 12509259
  34. [36]Fiala KA Suo Z Sloppy bypass of an abasic lesion catalyzed by a Y-family DNA polymerase J. Biol. Chem. 2007 282 8199 8206 17234630
  35. [37]Pusch CM Giddings I Scholz M Repair of degraded duplex DNA from prehistoric samples using Escherichia coli DNA polymerase I and T4 DNA ligase Nucleic Acids Res. 1998 26 857 859 9443981 PMC147318
  36. [38]Di Bernardo G Del Gaudio S Cammarota M Galderisi U Cascino A Cipollaro M Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains Nucleic Acids Res. 2002 30 e16 11842122 PMC100355
  37. [39]Rohland N Hofreiter M Comparison and optimization of ancient DNA extraction Biotechniques 2007 42 343 352 17390541
  38. [40]Maricic T Pääbo S Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands Biotechniques 2009 46 51 52, 54–57 19301622

Cited by

Sorted by citations

Authors
Heyn, P., Stenzel, U., Briggs, A. W., Kircher, M., Hofreiter, M., & Meyer, M.
Affiliations
  • Max Planck Institute for Evolutionary Anthropology (DE)
  • Max Planck Society (DE)
Corresponding author
  • Heyn, Patricia
  • Meyer, Matthias
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2010-06-28
Volume
38
Issue
16
Pages
e161-e161
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkq572
PMC
PMC2938203
OpenAlex
W2097501515
Cited by
56
References
2
Citations by year
2025: 2 · 2024: 2 · 2023: 1 · 2022: 1 · 2021: 2 · 2020: 4 · 2019: 2 · 2018: 2 · 2017: 4 · 2016: 2 · 2015: 3 · 2014: 10 · 2013: 3 · 2012: 9
FWCI
3.60
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion, other-oa)
  • Max Planck Institute for Plasma Physics (submittedVersion)
  • HighWire Press Open Archive (submittedVersion)
Funders
Max-Planck-Gesellschaft
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic diversity and population structure
Keywords
ancient DNA, endogenous DNA damage, single-molecule analysis, primer extension, paleogenomics
MeSH terms
DNA Damage, DNA-Directed DNA Polymerase, Fossils, Templates, Genetic, Polymerase Chain Reaction, Sequence Analysis, DNA, Genomics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review