2  Single stranded DNA Libraries

Figure 2.1: Homage to the best moustache ever (Salvador Dalí) by Nihan D. Dagtas. Based upon ‘Salvador Dalí’ by Philippe Halsman (1953) from wikiart.org. Used under the ‘Fair Use’ as defined on Wikiart and is used for educational purposes

Figure 2.2: Example of a smiley plot of a double stranded DNA library. Data taken from library VEL003.B0101 of (Andrades Valtueña et al. 2022). Damage data generated using DamageProfiler and plotted using R and tidyverse packages (Wickham et al. 2019).

This is an increasingly common ancient DNA plot that is you will see more often as single-stranded library construction protocols become more popular. 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). As with double-stranded ancient DNA libraries, the 5’ end will have the expect original C to T deamination curve. In contrast to the double stranded protocol, the 3’ of the molecule will also show the complementary C to T curve. You see the C to T deamination at both ends of the the molecule in this case, as during typical single-stranded library construction protcols (e.g. Gansauge et al. 2017) the entire DNA molecule is denatured, immobilised and then the complementary strand reconstructed - i.e., without any filling in or blunt ending. The highest frequency point of the curve can vary from 1% to ???% depending on the age and preservation of the sample.

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

Andrades Valtueña, Aida, Gunnar U Neumann, Maria A Spyrou, Lyazzat Musralina, Franziska Aron, Arman Beisenov, Andrey B Belinskiy, et al. 2022. “Stone Age Yersinia Pestis Genomes Shed Light on the Early Evolution, Diversity, and Ecology of Plague.” Proceedings of the National Academy of Sciences of the United States of America 119 (17): e2116722119. https://doi.org/10.1073/pnas.2116722119.

Gansauge, Marie-Theres, Tobias Gerber, Isabelle Glocke, Petra Korlevic, Laurin Lippik, Sarah Nagel, Lara Maria Riehl, Anna Schmidt, and Matthias Meyer. 2017. “Single-Stranded DNA Library Preparation from Highly Degraded DNA Using T4 DNA Ligase.” Nucleic Acids Research 45 (10): e79. https://doi.org/10.1093/nar/gkx033.

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.