Abstract
We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.
Highlights
We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones
If the amplified product has more than 1 X 1012 copies per ul, reduce the amount of template in the following Primer Extension step to ensure that not more than 2 X 1012 copies are added to the Primer Extension reaction
Representative Results: It is strongly recommended when starting out with the PEC protocol to perform first a capture reaction where a small amount of a 'positive control target sequence' (e.g. a ~100bp PCR product - 1 picogram is enough) is mixed in with the normal recommended amount of amplified 454 library template, and capture is performed with a single PEC primer designed to capture the positive control product
Summary
We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. To amplify the whole library, prepare the following PCR mix: Reagent μl per reaction 4. Quantify the purified amplified product as well as an aliquot of the unamplified library with qPCR (Meyer et al, 2007).
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call