1  Double stranded DNA libraries

Figure 1.1: Archibald by Tessa Zeibig

Figure 1.2: Example of a smiley plot of a double stranded DNA library. Data taken from library COD076E1bL1 (ERR1943600-ERR1943602) of (Star et al. 2017). Damage data generated using DamageProfiler and plotted using R and tidyverse packages (Wickham et al. 2019).

This is the ‘classical’ ancient DNA plot that you will see most often in palaeogenomics. You expect to see a smooth curve from the beginning of the read (position 1) to a flat line in the middle (e.g. positions 10-25 in mapDamage plots). At the 5’ end this will be indicated by the original C to T deamination, whereas the 3’ of the molecule will show the complementary G to A. You only see deamination the C to T (and complement G to A) at one end of the the molecule, as during typical double-stranded library construction protcols (Meyer and Kircher 2010) only one end of the single-ended overhangs of a DNA molecule is repaired by being ‘filled in’ (where the mis-reading of the deaminated C occurs). Overhangs at the other end of the molecule (which may also hold cytosine demination) are ‘blunt-ended’ by being trimmed off. Both fill-in and blunt-ending reactions are performed to allow ligation of next-generation-sequencing adapters and/or internal barcodes to both ends of the molecules. The highest frequency point of the curve can vary from 1% to 50% depending on the age and preservation of the sample.

If you get such a plot with smooth lines from ancient DNA double-stranded libraries, this is a good indication you have authentic ancient DNA!

Meyer, Matthias, and Martin Kircher. 2010. “Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing.” Cold Spring Harbor Protocols 2010 (6): db.prot5448. https://doi.org/10.1101/pdb.prot5448.

Star, Bastiaan, Sanne Boessenkool, Agata T Gondek, Elena A Nikulina, Anne Karin Hufthammer, Christophe Pampoulie, Halvor Knutsen, et al. 2017. “Ancient DNA Reveals the Arctic Origin of Viking Age Cod from Haithabu, Germany.” Proceedings of the National Academy of Sciences of the United States of America 114 (34): 9152–57. https://doi.org/10.1073/pnas.1710186114.

Wickham, Hadley, Mara Averick, Jennifer Bryan, Winston Chang, Lucy McGowan, Romain François, Garrett Grolemund, et al. 2019. “Welcome to the Tidyverse.” Journal of Open Source Software 4 (43): 1686. https://doi.org/10.21105/joss.01686.