In the present work extracts (200 µl) of ancient DNA contained between 4,720 and 375,120 molecules, i.e. 177 ancient templates were present in PCR reactions with the most dilute extract (using 7.5 µl) and thousands of molecules were available with the more concentrated extracts.
Phosphorylation is one of the many modifications possibly present at 3'-ends of ancient templates.
This suggests that the presence of deaminated cytosine residues reduces the number of hydrogen bonds between DNA strands and thereby likely favor a preferential denaturation of endogenous ancient templates, especially compared to longer, hence more thermostable, exogenous templates.
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.
In addition, the frequency of nucleotide misincorporation among reads shows a pattern typical of ancient templates, with an excess of type II over type I transitions (ratio typeII/typeI = 1.58; see Table S2, Additional File 3).
The labyrinthine Indian statistical system, created to meet the requirements of the colonial administration, continues to generate vast flows of statistics emerging from ancient templates that have not been sufficiently updated or reoriented to the fresh requirements of the times.
The extra hits identified from tSMS sequences were found to exhibit the nucleotide misincorporation pattern characteristic of ancient templates, with an excess of gaps opened at alignment termini (Additional file 1: Figure S7).
We should caution that DNA phosphatase treatments of ancient DNA extracts could be deleterious if second-generation sequencing methods and not tSMS were used as sequencing approaches, since the former require building ancient templates into DNA libraries through adapter ligation at phosphorylated 5'-ends.
Hence, we decided to test whether dephosphorylation of ancient DNA extracts could restore access to a significant fraction of ancient templates and therefore could improve the overall performance of tSMS by increasing either the overall number of sequences generated per channel or the fraction of endogenous sequences recovered.
Adding to the DNA damage induced nucleotide misincorporations that are typical of ancient templates, these specificities could limit our ability to map sequencing reads against modern reference genomes and therefore limit our ability to identify genuine endogenous tSMS reads, reducing the efficiency of shotgun sequencing.
