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single-stranded.qmd
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# Single stranded DNA Libraries
 from [wikiart.org](wikiart.org). Used under the 'Fair Use' as defined on Wikiart and is used for educational purposes](/assets/images/single-stranded/salvador.png){#fig-single-stranded-caricature height=400px}
```{r}
#| label: fig-single-stranded-smiley
#| fig-cap: Example of a smiley plot of a double stranded DNA library. Data taken from library VEL003.B0101 of [@Andrades_Valtuena2022-tq]. Damage data generated using [DamageProfiler](/intro.qmd) and plotted using R and tidyverse packages [@Wickham2019-ot].
#| echo: false
#| warning: false
#| error: false
library(readr)
library(tidyr)
library(dplyr)
library(ggplot2)
library(patchwork)
source("assets/src/R/style.r")
source("assets/src/R/damage-profiler.r")
type_colours <- type_colours_dp
raw_5p <- readr::read_tsv("assets/data/single-stranded/5p_freq_misincorporations.txt", comment = "#")
raw_3p <- readr::read_tsv("assets/data/single-stranded/3p_freq_misincorporations.txt", comment = "#")
long_5p <- pivot_longer_freqmisincorporat(raw_5p, reverse = FALSE, type_colours = type_colours)
long_3p <- pivot_longer_freqmisincorporat(raw_3p, reverse = TRUE, type_colours = type_colours)
smiley_5p <- plot_longer_freqmisincorporat(long_5p, reverse = FALSE, type_colours)
smiley_3p <- plot_longer_freqmisincorporat(long_3p, reverse = TRUE, type_colours)
smiley_5p + smiley_3p
```
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. @Gansauge2017-ge] 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!