Abstract
Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.
Highlights
The preservation of DNA in fossil specimens has opened new perspectives in evolutionary biology, providing access to genetic information from past individuals [1,2] and extinct species [3,4]
A complementary situation was observed at read ends where the last nucleotide position sequenced within reads was impoverished in adenine and enriched in cytosine while the first genomic location following sequencing reads was found to be enriched in guanine
The strong bias against templates starting with thymine residues, which we found being specific to AT libraries, could limit our ability to ligate ancient DNA templates, especially those starting with deaminated cytosines
Summary
The preservation of DNA in fossil specimens has opened new perspectives in evolutionary biology, providing access to genetic information from past individuals [1,2] and extinct species [3,4]. Base composition patterns have been critical for distinguishing among truly ancient sequence data and contamination [14,16]. One such procedure is based on the signature of the most prominent form of post-mortem DNA damage, namely cytosine deamination, which transforms native cytosines into uracils [17,18]. Replication over uracil residues during DNA library preparation and amplification generates spurious C T and G A misincorporations, especially at sequencing termini where deamination rates are increased by orders of magnitude due to the presence of single-stranded overhangs in ancient DNA fragments [16]
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