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Genome research2011; 21(10); 1705-1719; doi: 10.1101/gr.122747.111

True single-molecule DNA sequencing of a pleistocene horse bone.

Abstract: Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first "true single molecule sequencing" of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3' ends of ancient templates, indicating the presence of 3'-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second- and third-generation sequencing approaches.
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Publication Date: 2011-07-29 PubMed ID: 21803858PubMed Central: PMC3202287DOI: 10.1101/gr.122747.111Google Scholar: Lookup
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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 article presents the use of “true single molecule sequencing” for ancient DNA from a Pleistocene horse bone, showing it can provide a higher percentage of endogenous DNA sequences and offers an efficient way of studying ancient DNA extracts. The article also suggests that a combination of current second- and third-generation sequencing methods could enhance the sequencing of paleogenomes.

Introduction

  • The research focuses on using second-generation sequencing platforms for studying ancient DNA.
  • These platforms have been transformative in the study of past genomes, even from extinct species.
  • However, the process relies heavily on constructing libraries and amplification steps, which might alter the original DNA template composition.
  • This is especially an issue with ancient DNA, which has been subjected to significant damage over time.

Single Molecule Sequencing and its Application

  • The researchers employed “true single molecule sequencing” to assess ancient DNA.
  • DNA sequences from a Pleistocene horse bone, preserved in permafrost, were generated using the Helicos HeliScope and Illumina GAIIx platforms.
  • The sequences derived from the horse (endogenous DNA sequences) were found more frequently in Helicos data than in Illumina data, showing a distinct advantage of using the “true single molecule sequencing”.

Limitations of Existing Tools and Improvement Suggestion

  • Tools used for generating sequencing libraries often introduce biases, reducing the efficiency of the process and limiting the full exploration of the ancient DNA extracts’ molecular complexity.
  • The researchers found that making simple modifications to the standard Helicos DNA template preparation protocol could increase the horse DNA proportion in their sample by three times.

Analyzing Biases and Sequence Errors

  • The study compared Helicos-specific biases and sequence errors between modern and ancient DNA.
  • It revealed extensive cytosine deamination damage at the ancient DNA templates’ 3′ ends, indicating the presence of 3′-sequence overhangs.

Future Recommendations

  • The study concluded by suggesting a potential approach for sequencing paleogenomes using a combination of current second- and third-generation sequencing techniques.
  • This method could revolutionize the sequencing process of paleogenomes.

Cite This Article

APA
Orlando L, Ginolhac A, Raghavan M, Vilstrup J, Rasmussen M, Magnussen K, Steinmann KE, Kapranov P, Thompson JF, Zazula G, Froese D, Moltke I, Shapiro B, Hofreiter M, Al-Rasheid KA, Gilbert MT, Willerslev E. (2011). True single-molecule DNA sequencing of a pleistocene horse bone. Genome Res, 21(10), 1705-1719. https://doi.org/10.1101/gr.122747.111

Publication

Genome research
ISSN: 1549-5469
NlmUniqueID: 9518021
Country: United States
Language: English
Volume: 21
Issue: 10
Pages: 1705-1719

Researcher Affiliations

Orlando, Ludovic
  • Centre for GeoGenetics, Natural History Museum of Denmark, Copenhagen University, Copenhagen DK-1350, Denmark. Lorlando@snm.ku.dk
Ginolhac, Aurelien
    Raghavan, Maanasa
      Vilstrup, Julia
        Rasmussen, Morten
          Magnussen, Kim
            Steinmann, Kathleen E
              Kapranov, Philipp
                Thompson, John F
                  Zazula, Grant
                    Froese, Duane
                      Moltke, Ida
                        Shapiro, Beth
                          Hofreiter, Michael
                            Al-Rasheid, Khaled A S
                              Gilbert, M Thomas P
                                Willerslev, Eske

                                  MeSH Terms

                                  • Animals
                                  • Bone and Bones / chemistry
                                  • Chromosome Mapping
                                  • DNA / chemistry
                                  • DNA / isolation & purification
                                  • DNA Damage
                                  • DNA Fragmentation
                                  • Fossils
                                  • High-Throughput Nucleotide Sequencing / instrumentation
                                  • High-Throughput Nucleotide Sequencing / methods
                                  • Horses / genetics
                                  • Sequence Analysis, DNA / instrumentation
                                  • Sequence Analysis, DNA / methods

                                  Grant Funding

                                  • R44 HG005279 / NHGRI NIH HHS
                                  • R44 HG005279-02 / NHGRI NIH HHS

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