Article (Scientific journals)
True single-molecule DNA sequencing of a pleistocene horse bone.
Orlando, Ludovic; GINOLHAC, Aurélien; Raghavan, Maanasa et al.
2011In Genome Research, 21 (10), p. 1705-19
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Keywords :
Animals; Bone and Bones/chemistry; Chromosome Mapping; DNA/chemistry/isolation & purification; DNA Damage; DNA Fragmentation; Fossils; High-Throughput Nucleotide Sequencing/instrumentation/methods; Horses/genetics; Sequence Analysis, DNA/instrumentation/methods
Abstract :
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Orlando, Ludovic
GINOLHAC, Aurélien  ;  University of Copenhagen > Centre for Geogenetics
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
More authors (7 more) Less
External co-authors :
yes
Language :
English
Title :
True single-molecule DNA sequencing of a pleistocene horse bone.
Publication date :
2011
Journal title :
Genome Research
ISSN :
1088-9051
eISSN :
1549-5469
Publisher :
Cold Spring Harbor Laboratory Press, United States - New York
Volume :
21
Issue :
10
Pages :
1705-19
Peer reviewed :
Peer Reviewed verified by ORBi
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