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Improving the performance
of True Molecule Sequencing for ancient DNA
Aurélien Ginolhac1, Julia Vilstrup1, Jesper Stenderup1, Morten Rasmussen1, Mathias Stiller2, Beth Shapiro2,
Grant Zazula3, Duane Froese4, Kathleen E. Steinmann5,6, John F. Thompson5,7, Khaled A.S. AL-Rasheid8,
Thomas M.P. Gilbert1, Eske Willerslev1 and Ludovic Orlando1
1 Centre for GeoGenetics, Natural History Museum of Denmark, Copenhagen University, 5-7, Øster Voldgade, Københavns 1350, Denmark, 2 Department of Biology, The Pennsylvania State University, 326 Mueller Lab, University Park, PA
16802, USA, 3 Department of Tourism and Culture, Government of Yukon, PO Box 2703 L2A, Whitehorse, Yukon Territory Y1A, 2C6, Canada, 4 Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, Alberta T6G 2E3,
Canada, 5 Applications, Methods and Collaborations, Helicos BioSciences, One Kendall Square Bldg 200LL, Cambridge, MA 02139, USA, 6 Present address: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA
021423, USA, 7 Present address: NABsys Inc, Providence, RI 0290, USA, 8 Zoology Department, College of Science King Saud University, P.O. Box, 2455, Riyadh 11451, Saudi Arabia
Contacts: [email protected], [email protected]
Background
Ancient DNA has been shown to survive in fossil material however, post-mortem DNA damage
reactions, which fragment the DNA backbone into short pieces and generate hydrolytic and oxidative
base derivatives, often limit the amount of DNA templates preserved. Since extraction methods are
destructive and samples rare, optimizing the number of recovered genuine reads is at utmost
importance.
Aims
In this study, we investigated the impact of phosphate groups at 3’-ends that could remain after
breaks in DNA backbones. Those groups prevent their sequencing using the HeliScope platform since
they must be poly-A tailed on their free 3’-OH (Fig 1, B). Furthermore, regarding the short size of
ancient templates, two denaturation temperatures were evaluated. Extracts from Pleistocene horse
bones were obtained in duplicates for: 1) a specimen with an infinite radiocarbon date CA1 and CA2;
2) a specimen dated 13,389BP, TP1 and TP2. Remaining pellets from the second specimen were reextracted and are referred to TP1RE and TP2RE respectively. DNA extracts for CA1 and CA2 were
treated independently using 2 protocols 1) with 95ºC for denaturation; 2) 80ºC for denaturation.
DNA extracts from TP1, TP1RE, TP2 and TP2RE were treated independently using protocols 1) and
and 2) plus one using the Antartica DNA phosphatase and 80ºC for denaturation. The mapping
procedure was optimized following recent recommended settings for ancient DNA [1].
Results
We found that treating ancient DNA extracts with DNA phosphatase improved the amount of
endogenous sequence information recovered per sequencing channel by up to 6.8-fold (Table 1),
while still providing molecular signatures of endogenous ancient DNA damage, including cytosine
deamination (Fig2.) and fragmentation by depurination (Fig 3). Additionally, we confirmed the
existence of molecular preservation niches in large bone crystals [3] from which DNA could be
preferentially extracted and that mild denaturation temperature favored ancient templates (Fig 2).
Fig 1. Remaining phosphate group at 3’-ends prevent Helicos sequencing
A. DNA molecules with phosphate groups are not substrate for the Terminal Transferase.
B. Poly-A tailing was performed only for the molecule with a 3’-OH.
C. Capture on the Helicos HeliScope flow cell, the first molecule is lost at this stage.
D. Helicos sequencing starts will filling with Ts (in black), then a blocker
(in blue, A, G or C). Effective sequencing starts afterwards (Zs in green).
Table 1. Ratio of numbers of reads recovered and percentage of endogenous content
80ºC described experiments without phosphatase treatment and a denaturation temperature of 80ºC.
P, 80ºC referred to phosphatase treatment experiments and denaturation temperature set to 80ºC.
%End. 95ºC refers to the percentage of endogenous horse reads obtained using a denaturation
temperature of 95ºC for the 6 DNA extracts.
TP1
TP1 re-extraction
TP2
TP2 re-extraction
Fig 2. Cumulative guanine to adenine misincorporation rates per read position
This class of mismatch derives from the post-mortem deamination of cytosine residues and
can be taken as a proxy for post-mortem DNA damage.
Fig 3. Base composition at the 5’-end of tSMS reads and preceding genomic regions.
After DNA phosphatase treatment, position -2 was found to be enriched in cytosine residues, in
agreement with the model of DNA fragmentation through depurination [2].
Position -1, following DNA phosphatase it was found to be enriched in pyrimidines. This position
was previously shown to preferentially consist of guanine residues [3].
Position +1, shown an increase in Cs indicating lower levels of damages (Fig 2, TP1 and TP2).
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 [4].
Acknowledgments
We thank Tina Brand and the laboratory technicians at the Danish High-throughput DNA Sequencing Centre for technical assistance; Anders Krogh,
Stinus Lindgreen, Mikkel Schubert, Maanasa Raghavan. This work was supported by the Danish Council for Independent Research, Natural Sciences
(FNU); the Danish National Research Foundation; a Marie-Curie Career Integration Grant CIG- 293845; the National Science Foundation ARC0909456; the Searle Scholars Program; King Saud University Distinguished Scientist Fellowship Program (DSFP). This project was supported in part
by American Recovery and Reinvestment Act (ARRA) funds through grant number RC2 HG005598 to JFT from the National Human Genome Research
Institute, National Institutes of Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views
of NHGRI.
References.
[1] Schubert et al. Improving ancient DNA mapping against modern
reference genomes. BMC Genomics 2012, 13:178.
[2] Briggs et al. Removal of deaminated cytosines and detection of in
vivo methylation in ancient DNA. Nucleic Acids Res 2010, 38:e87.
[3] Orlando et al. True Single-molecule DNA sequencing of a
Pleistocene horse bone. Genome Res 2011, 21:1705-1719
[4] Ginolhac et al. Improving the performance of True Single Molecule
Sequencing for ancient DNA. BMC Genomics 2012, 13:177.