How many clones need to be sequenced from a single forensic or ancient DNA sample in order to determine a reliable consensus sequence?
Date
2005
Authors
Bower, M.
Spencer, M.
Matsumura, S.
Nisbet, E.
Howe, C.
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Advisors
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Type:
Journal article
Citation
Nucleic Acids Research (NAR), 2005; 33(8):2549-2556
Statement of Responsibility
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Abstract
Forensic and ancient DNA (aDNA) extracts aremixtures of endogenous aDNA, existing in moreor less damaged state, and contaminant DNA. Toobtain the true aDNA sequence, it is not sufficientto 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. Onlybacterial cloning can elucidate the components ofthis mixture. We calculate the number of clonesthat need to be sampled (for various mixture ratios)in order to be confident (at various levels of confidence)to have identified the major component. Wedemonstrate that to be .95% confident of identifyingthe most abundant sequence present at 70%in the ancient sample, 20 clones must be sampled.We make recommendations and offer a free-accessweb-based program, which constructs the mostreliable consensus sequence from the users inputclone sequences and analyses the confidencelimits for each nucleotide position and for thewhole consensus sequence. Accepted authenticationmethods must be employed in order to assessthe authenticity and endogeneity of the resultingconsensus sequences (e.g. quantification andreplication by another laboratory, blind testing,amelogenin sex versus morphological sex, the effectiveuse of controls, etc.) and determine whether theyare indeed aDNA.