TY - JOUR
T1 - True single-molecule DNA sequencing of a pleistocene horse bone
AU - Orlando, Ludovic Antoine Alexandre
AU - Ginolhac, Aurélien
AU - Raghavan, Maanasa
AU - Mouatt, Julia Thidamarth Vilstrup
AU - Rasmussen, Morten
AU - Magnussen, Kim
AU - Steinmann, Kathleen E.
AU - Kapranov, Philipp
AU - Thompson, John F.
AU - Zazula, Grant
AU - Froese, Duane
AU - Moltke, Ida
AU - Shapiro, Beth
AU - Hofreiter, Michael
AU - Al-Rasheid, Khaled A. S.
AU - Gilbert, M. Thomas P.
AU - Willerslev, Eske
PY - 2011
Y1 - 2011
N2 - 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 115.9Mb and 76.9Mb 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 3-fold. 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.
AB - 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 115.9Mb and 76.9Mb 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 3-fold. 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.
U2 - 10.1101/gr.122747.111
DO - 10.1101/gr.122747.111
M3 - Journal article
C2 - 21803858
VL - 21
SP - 1705
EP - 1719
JO - Genome Research
JF - Genome Research
SN - 1088-9051
IS - 10
ER -