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Nucleic Acids Research 2005 33(8):2549-2556; doi:10.1093/nar/gki550
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Published online 3 May 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

How many clones need to be sequenced from a single forensic or ancient DNA sample in order to determine a reliable consensus sequence?

Mim A. Bower*, Matthew Spencer1, Shuichi Matsumura, R. Ellen R. Nisbet2 and Christopher J. Howe2

Archaeogenetics Laboratory, McDonald Institute for Archaeological Research, University of Cambridge Downing Street, Cambridge CB2 3ER, UK 1Department of Mathematics and Statistics, Dalhousie University Halifax, Nova Scotia, B3H 3J5, Canada 2Department of Biochemistry, University of Cambridge Tennis Court Road, Cambridge CB2 1QW, UK

*To whom correspondence should be addressed. Tel: +44 1223 339297; Fax: +44 1223 339285; Email: mab1004{at}cam.ac.uk

Received March 10, 2005. Revised April 7, 2005. Accepted April 15, 2005.

Forensic and ancient DNA (aDNA) extracts are mixtures of endogenous aDNA, existing in more or less damaged state, and contaminant DNA. To obtain the true aDNA sequence, it is not sufficient to generate a single direct sequence of the mixture, even where the authentic aDNA is the most abundant (e.g. 25% or more) in the component mixture. Only bacterial cloning can elucidate the components of this mixture. We calculate the number of clones that need to be sampled (for various mixture ratios) in order to be confident (at various levels of confidence) to have identified the major component. We demonstrate that to be >95% confident of identifying the most abundant sequence present at 70% in the ancient sample, 20 clones must be sampled. We make recommendations and offer a free-access web-based program, which constructs the most reliable consensus sequence from the user's input clone sequences and analyses the confidence limits for each nucleotide position and for the whole consensus sequence. Accepted authentication methods must be employed in order to assess the authenticity and endogeneity of the resulting consensus sequences (e.g. quantification and replication by another laboratory, blind testing, amelogenin sex versus morphological sex, the effective use of controls, etc.) and determine whether they are indeed aDNA.


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