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BMC genomics2012; 13; 177; doi: 10.1186/1471-2164-13-177

Improving the performance of true single molecule sequencing for ancient DNA.

Abstract: Second-generation sequencing technologies have revolutionized our ability to recover genetic information from the past, allowing the characterization of the first complete genomes from past individuals and extinct species. Recently, third generation Helicos sequencing platforms, which perform true Single-Molecule DNA Sequencing (tSMS), have shown great potential for sequencing DNA molecules from Pleistocene fossils. Here, we aim at improving even further the performance of tSMS for ancient DNA by testing two novel tSMS template preparation methods for Pleistocene bone fossils, namely oligonucleotide spiking and treatment with DNA phosphatase. Results: We found that a significantly larger fraction of the horse genome could be covered following oligonucleotide spiking however not reproducibly and at the cost of extra post-sequencing filtering procedures and skewed %GC content. In contrast, we showed that treating ancient DNA extracts with DNA phosphatase improved the amount of endogenous sequence information recovered per sequencing channel by up to 3.3-fold, while still providing molecular signatures of endogenous ancient DNA damage, including cytosine deamination and fragmentation by depurination. Additionally, we confirmed the existence of molecular preservation niches in large bone crystals from which DNA could be preferentially extracted. Conclusions: We propose DNA phosphatase treatment as a mechanism to increase sequence coverage of ancient genomes when using Helicos tSMS as a sequencing platform. Together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, this simple preparation procedure can improve overall Helicos tSMS performance when damaged DNA templates are targeted.
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Publication Date: 2012-05-10 PubMed ID: 22574620PubMed Central: PMC3430569DOI: 10.1186/1471-2164-13-177Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • American Recovery and Reinvestment Act
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • Non-U.S. Gov't
  • Research Support
  • U.S. Gov't
  • Non-P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research explores ways to improve true Single-Molecule DNA Sequencing (tSMS) for ancient DNA by testing two methods of preparation for Pleistocene bone fossils. It found success with DNA phosphatase and highlighted potential molecular preservation techniques in large bone crystals.

Introduction to the Study

  • The research was carried out as a part of the effort to enhance the potential of third-generation Helicos sequencing platforms in the sequencing of Pleistocene fossil DNA molecules.
  • Such DNA sequencing has radically improved our capacity to get genetic information from historical time. This breakthrough has enabled us to characterize the complete genomes of extinct species and individuals from the past.

Methods and Approaches Adapted in the Study

  • The study tests two new template preparation techniques for ancient DNA: oligonucleotide spiking and DNA phosphatase treatment applied to Pleistocene bone fossils.
  • It tries to find the most efficient method that can enhance the performance of tSMS.

Key Findings of the Study

  • The application of oligonucleotide spiking allowed for a more significant fraction of the horse genome to be covered but not reproducibly. It also required additional post-sequencing filtering processes and resulted in skewed % GC content.
  • DNA phosphatase treatment on ancient DNA extracts improved the recovery rate of endogenous sequence data per sequencing channel by up to 3.3 times. It also helped provide molecular signatures of endogenous ancient DNA damage, such as depurination fragmentation and cytosine deamination.
  • The study confirmed that there exist molecular preservation niches in substantial bone crystals from where DNA could be preferably extracted.

Conclusions of the Study

  • The study endorses DNA phosphatase treatment as a beneficial method to raise sequence coverage of ancient genomes when Helicos tSMS is used as a sequencing platform.
  • This straightforward preparation procedure, together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, can enhance the Helicos tSMS performance when damaged DNA templates are targeted.

Cite This Article

APA
Ginolhac A, Vilstrup J, Stenderup J, Rasmussen M, Stiller M, Shapiro B, Zazula G, Froese D, Steinmann KE, Thompson JF, Al-Rasheid KA, Gilbert TM, Willerslev E, Orlando L. (2012). Improving the performance of true single molecule sequencing for ancient DNA. BMC Genomics, 13, 177. https://doi.org/10.1186/1471-2164-13-177

Publication

BMC genomics
ISSN: 1471-2164
NlmUniqueID: 100965258
Country: England
Language: English
Volume: 13
Pages: 177

Researcher Affiliations

Ginolhac, Aurelien
  • Centre for GeoGenetics, Natural History Museum of Denmark, Copenhagen University, 5-7 Øster Voldgade, København, 1350, Denmark.
Vilstrup, Julia
    Stenderup, Jesper
      Rasmussen, Morten
        Stiller, Mathias
          Shapiro, Beth
            Zazula, Grant
              Froese, Duane
                Steinmann, Kathleen E
                  Thompson, John F
                    Al-Rasheid, Khaled A S
                      Gilbert, Thomas M P
                        Willerslev, Eske
                          Orlando, Ludovic

                            MeSH Terms

                            • Animals
                            • Base Composition / genetics
                            • Base Sequence
                            • DNA / genetics
                            • DNA, Mitochondrial / genetics
                            • Fossils
                            • Genome / genetics
                            • Horses / genetics
                            • Nucleotidases / metabolism
                            • Nucleotides / genetics
                            • Phylogeny
                            • Sequence Analysis, DNA / methods
                            • Sequence Analysis, DNA / standards

                            Grant Funding

                            • RC2 HG005598 / NHGRI NIH HHS

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