```
# download Trident v1.4.0.3 binary
wget https://github.com/poseidon-framework/poseidon-hs/releases/download/v1.4.0.3/trident-Linux
# rename to trident
mv trident-Linux trident
# make it executable
chmod +x trident
# run it
./trident -h
```

# 8 Dimensionality reduction using PCA and MDS

## 8.1 Theory

### 8.1.1 Rationale

The datasets used in human ancient DNA analysis are often extremely multidimensional, often including data from thousands of individuals, across hundreds of thousands (or millions!) of single nucleotide polymrphisms (SNPs) (Mallick et al. 2023). Even when choosing to summarise this genome-wide information to single statistics of genetic similarity (e.g. with Outgroup F3), a similarity matrix across individuals can become very large when comparing across hundreds of individuals. As the name implies, dimensionality reduction methods can reduce the number of dimensions in the underlying data, while also aiming to minimise the loss of information. The two such methods we will focus on in this tutorial are **Principal Component Analysis (PCA)** and **Multi-Dimensional Scaling (MDS)**. Both methods reveal structure within the dataset, and part of that structure is due to shared population history between individuals/populations. It is for that reason that both these methods are indispensible parts of an archaeogeneticist’s toolkit.

### 8.1.2 Introduction to dimension reduction

When using either of these methods, we are essentially representing the data on a new set of **orthogonal** axes, with its origin in the center of the data. In PCA we typically use the original data for this transformation (i.e. the genotype matrix), and attempt to find the axes that capture the most variation among the samples. A covariance matrix (i.e. a similarity matrix) is often calculated and used as a useful intermediate step in PCA. Instead, in MDS we start with a pairwise distance matrix (typically a matrix of 1-F3), and attempt to find a spatial representation that best captures the distances between points.

The results of both of these methods are (usually) a 2 dimensional plot in which the distances between individual points roughly correlates to the genetic distance between these individuals. Therefore, genetically similar individuals will be plotted close to one another, and further away from individuals that are more genetically dissimilar.

### 8.1.3 The problem with missing data

A recurring issue when analysing ancient DNA is the high degree of missing data (i.e. missingness). We often apply a minimum coverage filter to our datasets: A generally accepted rule-of-thumb for the 1240K dataset is a minimum of 15 000 covered (i.e. non-missing) SNPs. Another way to express this cutoff is to say that we will “happily” analyse data that is missing a genotype call in **98.8%** of all SNPs in the dataset! So how does this high rate of missingness affect MDS and PCA?

#### 8.1.3.1 MDS

Missingness does not affect MDS as adversely as it does PCA, on account of the use of a pairwise distance matrix of 1-F3. This matrix will only have missing values in cases where there is no overlapping coverage between two individuals/populations used in an F3 statistic. Instead, the issue with MDS is that all F3 statistics are treated as equally reliable, regardless of their associated error bar.

#### 8.1.3.2 PCA

Unlike MDS, PCA is severely affected by missing data. During the rescaling of the data around its own mean values, missing data is “filled-in” to the mean value (mean imputation). This can cause points to shift towards the origin by a distance relative to the degree of missingness.

Below is a plot of the results of PCA on a dataset of differnt worldwide populations (Reich et al. 2012). In an attempt to limit the effects of colonial admixture on the studied Native American populations, the authors masked parts of the genomes of Native Americans that matched the European or African populations in their dataset, replacing those genotypes with missing data.

*Nature*488 (7411): 370–74.

As you can see, individuals whose genotypes were masked are shifted towards the plot’s origin. So how can we use PCA with ancient samples that have high degrees of missingness? The answer is by using a **L**east **Sq**uares **Project**ion, a.k.a. `lsqproject`

!

### 8.1.4 Projection

The idea of projection is simple, and applies similarly to both PCA and MDS. In PCA, you use a subset of the dataset to calculate your axes of variation, and then apply the resulting transformation to additional data, thus projecting them onto those axes. The important detail is that the variation between projected individuals is not taken into account when deciding which the axes of maximal variation are. Similarly, in MDS you project points to the MDS space based on their distances to the points that constructed the space, disregarding the distances of the projected points to one another.

Below is a PCA plot calculated on present-day West Eurasian populations together with some Mesolithic hunter-gatherer individuals (in light brown). In the right side plot, the ancient individuals have been included in the calculation of the principal components, while in the left side they are projected on the principal components of the present-day West Eurasians.

There are two things to note here:

- First, comparing the placement of Eastern European hunter-gatherers (brown right-facing triangles) between the two plots, you can see that projecting these individuals does indeed provide results that are not affected by mean imputation, and thus are not shifted towards the origin.
- Secondly, if you compare the positions of the Western European hunter-gatherers (brown half-filled circles), you will notice that projection causes these individuals to be plotted closer to present-day populations.

The degree of missingness in the Western European hunter-gatherers (WHG) is relatively low, and hence the shift in their placement between the two plots is not the result of mean imputation. Instead, when projected the WHG illustrate the effects of **shrinkage**.

### 8.1.5 Shrinkage

Shrinkage comes in two flavours:

The kind of shrinkage you saw with the WHGs above, is pretty intuitive. When projecting populations on axes of variation that do not capture all the variation of the projected populations, they will appear as if they have less variation than reality. This translates to the points “shrinking” towards the origin slightly. that is to say, because the WHG individuals come from a population that harboured far more genetic variation than is present within present-day West Eurasian populations, much of their true variation is “hidden” when projecting them.

The second kind of shrinkage (a.k.a. projection bias) arises because “samples used to calculate the PC axes”stretch” the axes” (from the smartpca documentation). This problem is exacerbated in datasets where the number of markers far exceeds the number of samples used for PC calculation. This is often the case in human population genomics.

While the first shrinkage flavour can be argued to be a feature of PCA, projection bias can be a problem when trying to compare present-day populations to projected ancient populations. A demonstration of the effects of shrinkage can be seen below:

Shrinkage can be corrected by scaling the eigenvectors of the projected and/or non-projected individuals to bring them more in line with one another. Below is the same dataset as above, but ran through smartpca with the parameter `shrinkmode: YES`

:

As a note of caution, `shrinkmode: Yes`

increases the runtime greatly. An alternative would be to identify specific present-day populations that are of interest for the ancient-to-modern comparison, and project those as well. So, for example, if we were to compare Iron Age individuals from Germany with present-day individuals from Germany, then we could decide to take out some or all present-day Germans and project those as well. That would make them fully comparable.

## 8.2 Practice

### 8.2.1 Preparation

#### 8.2.1.1 Get trident

Trident is a Poseidon framework data management tool. It enables downloading Poseidon packages (genomic datasets, usually including ancient individuals, comprising genome-wide SNP data) from the Poseidon server, as well as creating and manipulating such packages.

trident for Linux:

trident for MacOS:

```
# download Trident v1.4.0.3 binary
curl -LO https://github.com/poseidon-framework/poseidon-hs/releases/download/v1.4.0.3/trident-macOS
# rename to trident
mv trident-macOS trident
# make it executable
chmod +x trident
# run it
./trident -h
```

trident for Windows:

Download trident-Windows.exe file

#### 8.2.1.2 Prepare practise dataset:

Here, we are downloading packages (listed in pca_mds_working/exampleData.fetchFile.txt) file from the Poseidon server into the scratch/poseidon-repository directory. The datasets come from the following publications: Patterson et al. 2012, Lazaridis et al. 2014, Raghavan et al. 2014, and Jeong et al. 2019

```
mkdir -p scratch/poseidon-repository
# This will take a few seconds to pull the data from the server
./trident fetch -d scratch/poseidon-repository --fetchFile "pca_mds_working/exampleData.fetchFile.txt"
```

```
# Check composition of one of the downloaded packages
ls scratch/poseidon-repository/2014_LazaridisNature-4.0.2
```

```
# List all groups (populations) comprised by the downloaded packages
./trident list --groups -d scratch/poseidon-repository/
```

```
# Summarize information about the downloaded packages
./trident summarise -d scratch/poseidon-repository
```

```
# Choose populations for the analysis (list for this exercise in "exampleData.forgeFile.txt"")
head pca_mds_working/exampleData.forgeFile.txt
```

```
# Count the number of listed populations to include
wc -l pca_mds_working/exampleData.forgeFile.txt
```

```
# Create (forge) a new repository with chosen groups from the downloaded packages
./trident forge \
-d scratch/poseidon-repository \
-o scratch/forged_package \
-n PCA_package_1 \
--forgeFile pca_mds_working/exampleData.forgeFile.txt \
--outFormat EIGENSTRAT
```

The created repository comprises genomic data for: 111 modern Eurasian populations, 6 modern Native American populations, and 1 Upper Palaeolithic Siberian individual MA-1 (“Mal’ta”).

### 8.2.2 Run PCA

```
# Prepare parameter file for the smartpca run
mkdir -p scratch/smartpca_runs/poplist1 scratch/smartpca_runs/poplist2/
cat <<EOF > scratch/smartpca_runs/poplist1/parameters.par
genotypename: scratch/forged_package/PCA_package_1.geno ## Genotype data
snpname: scratch/forged_package/PCA_package_1.snp ## SNP information
indivname: scratch/forged_package/PCA_package_1.ind ## Individual information
evecoutname: scratch/smartpca_runs/poplist1/PCA_poplist1.evec ## Eigenvectors
evaloutname: scratch/smartpca_runs/poplist1/PCA_poplist1.eval ## Eigenvalues
poplistname: pca_mds_working/PCA_poplists/PCA_poplist1.txt
lsqproject: YES ## Project individuals not included in PC calculation onto the PCs
outliermode: 2 ## Turns off automatic outlier removal.
numoutevec: 4 ## The number of eigenvectors to print per sample. Default is 10.
EOF
```

So prepared parameter file will cause smartpca to estimate PCs using only the individuals from the populations listed in PCA_poplist1.txt and project all the remaining individuals onto those estimated PCs.

```
# Run smartpca
smartpca -p scratch/smartpca_runs/poplist1/parameters.par
```

```
# Inspect the output files
ls scratch/smartpca_runs/poplist1/
head scratch/smartpca_runs/poplist1/PCA_poplist1.evec
```

Adding populations (Native Americans)

```
# Look into other provided poplists
wc -l pca_mds_working/PCA_poplists/*
diff -y --suppress-common-lines pca_mds_working/PCA_poplists/PCA_poplist1.txt pca_mds_working/PCA_poplists/PCA_poplist2.txt
```

```
# Replace poplist1 with poplist2 in the smartpca parameter file
sed 's/poplist1/poplist2/g' scratch/smartpca_runs/poplist1/parameters.par > scratch/smartpca_runs/poplist2/parameters.par
cat scratch/smartpca_runs/poplist2/parameters.par
```

```
# Rerun smartpca using poplist2 (additional populations)
smartpca -p scratch/smartpca_runs/poplist2/parameters.par
# Inspect smartpca output
ls scratch/smartpca_runs/poplist2/
```

Skipping projection (running smartpca without a poplist)

```
mkdir -p scratch/smartpca_runs/all_pops
head -n 7 scratch/smartpca_runs/poplist1/parameters.par | sed 's/poplist1/all_pops/g' > scratch/smartpca_runs/all_pops/parameters.par
tail -n 2 scratch/smartpca_runs/poplist1/parameters.par >> scratch/smartpca_runs/all_pops/parameters.par
echo "maxpops: 200" >> scratch/smartpca_runs/all_pops/parameters.par
echo "fastmode: YES" >> scratch/smartpca_runs/all_pops/parameters.par
cat scratch/smartpca_runs/all_pops/parameters.par
```

```
## Runtime of about 2 minutes
smartpca -p scratch/smartpca_runs/all_pops/parameters.par
ls scratch/smartpca_runs/all_pops/
```

**As a result we have three PCAs:**

data used in PC estimation | data projected | |
---|---|---|

PCA_poplist1 | Eurasians | Native Americans, Mal’ta |

PCA_poplist2 | Eurasians, Native Americans | Mal’ta |

PCA_all_pops | Eurasians, Native Americans, Mal’ta |

### 8.2.3 Plot PCA

```
library(tidyverse)
if(!require('remotes')) install.packages('remotes')
if (!require('janno')) remotes::install_github('poseidon-framework/janno')
## Load in poplist data
## poplist1 -- Eurasian populations
<- readr::read_tsv("pca_mds_working/PCA_poplists/PCA_poplist1.txt", col_names = "Pops", col_types = 'c')
poplist1 ## poplist2 -- Eurasian populations + 6 Native American populations
<- readr::read_tsv("pca_mds_working/PCA_poplists/PCA_poplist2.txt", col_names = "Pops", col_types = 'c')
poplist2
## Load in eigenvector data
<- readr::read_fwf("scratch/smartpca_runs/poplist1/PCA_poplist1.evec", col_positions=readr::fwf_widths(c(20,11,12,12,12,19), col_names = c("Ind","PC1","PC2","PC3","PC4","Pop")), col_types = 'cnnnnc', comment="#")
PCA_poplist1_ev <- readr::read_fwf("scratch/smartpca_runs/poplist2/PCA_poplist2.evec", col_positions=readr::fwf_widths(c(20,11,12,12,12,19), col_names = c("Ind","PC1","PC2","PC3","PC4","Pop")), col_types = 'cnnnnc', comment="#")
PCA_poplist2_ev <- readr::read_fwf("scratch/smartpca_runs/all_pops/PCA_all_pops.evec", col_positions=readr::fwf_widths(c(20,11,12,12,12,19), col_names = c("Ind","PC1","PC2","PC3","PC4","Pop")), col_types = 'cnnnnc', comment="#")
PCA_all_pops_ev
## Finally, we load in the metadata from the forged package annotation file (janno). Here, we keep only the individual Ids, country and their Lat/Lon position.
<-janno::read_janno("scratch/forged_package/PCA_package_1.janno", to_janno=F)%>% select(Poseidon_ID, Latitude, Longitude, Country) %>% mutate(Longitude=as.double(Longitude), Latitude=as.double(Latitude))
metadata
## Finally, we add the Lat/Lon information to our datasets
<- left_join(PCA_poplist1_ev, metadata, by=c("Ind"="Poseidon_ID")) %>% mutate(Country=as.factor(Country))
PCA_poplist1_ev <- left_join(PCA_poplist2_ev, metadata, by=c("Ind"="Poseidon_ID")) %>% mutate(Country=as.factor(Country))
PCA_poplist2_ev <- left_join(PCA_all_pops_ev, metadata, by=c("Ind"="Poseidon_ID")) %>% mutate(Country=as.factor(Country)) PCA_all_pops_ev
```

```
## First we subset the dataset to only the populations in the poplist
<- PCA_poplist1_ev %>% filter(Pop %in% poplist1$Pops)
moderns_pl1
<- ggplot() +
p coord_equal(xlim=c(-0.05,0.05),ylim=c(-0.05,0.15)) +
theme_minimal()
+ geom_point(
p data=moderns_pl1, ##The input data for plotting
aes(x=PC1, y=PC2) ## Define the x and y axis
)
```

```
## We can see how genetic similarity depends on geographical location by colouring the poins by longitude or latitude
<- p +
Lon_plot geom_point(data=moderns_pl1, aes(x=PC1, y=PC2, col=Longitude)) ## Here we also define the colour of the points based on a variable
<- p +
Lat_plot geom_point(data=moderns_pl1, aes(x=PC1, y=PC2, col=Latitude))
::grid.arrange(Lon_plot, Lat_plot, ncol=2) gridExtra
```

```
## As the orientation (+/-) of PC coordinates sometimes can change between runs of PCA, we use this code to ensure the same "orientation" for all users and thus enable making comparisons.
<- moderns_pl1 %>% select(Ind, PC1, PC2) %>% filter(Ind %in% c("HGDP00607", "Sir50"))
corner_inds_pl1 if (corner_inds_pl1$PC1[1] > corner_inds_pl1$PC1[2]) { PCA_poplist1_ev <- PCA_poplist1_ev %>% mutate(PC1=-PC1)}
if (corner_inds_pl1$PC2[1] > corner_inds_pl1$PC2[2]) { PCA_poplist1_ev <- PCA_poplist1_ev %>% mutate(PC2=-PC2)}
<- PCA_poplist1_ev %>% filter(Pop %in% poplist1$Pops)
moderns_pl1
## Let's now colour the points by country.
<- p +
PCA_plot_1 geom_point(data=moderns_pl1,
aes(x=PC1, y=PC2, col=Country),
alpha=0.5 ## Makes points semi transparent (so that aggregations of points are visible).
) PCA_plot_1
```

```
## Now, let's see where Mal'ta individual got projected
+
PCA_plot_1 geom_point(
data=PCA_poplist1_ev %>% filter(Ind=="MA1.SG"), ## Extract MA1 from the entire dataset
aes(x=PC1, y=PC2), ## Set the x and y axes for this set of points
pch=17 ## Change shape of point to solid triangle
)ggsave("scratch/PCA_plot_1.png")
```

PCA with Native American populations added to the analysis

```
## First we reorient the PCA
<- PCA_poplist2_ev %>% select(Ind, PC1, PC2) %>% filter(Ind %in% c("HGDP00607", "Sir50"))
corner_inds_pl2 if (corner_inds_pl2$PC1[1] > corner_inds_pl2$PC1[2]) { PCA_poplist2_ev <- PCA_poplist2_ev %>% mutate(PC1=-PC1)}
if (corner_inds_pl2$PC2[1] > corner_inds_pl2$PC2[2]) { PCA_poplist2_ev <- PCA_poplist2_ev %>% mutate(PC2=-PC2)}
<- PCA_poplist2_ev %>% filter(Pop %in% poplist2$Pops)
moderns_pl2
## Then we plot the output
<- ggplot() +
PCA_plot_2 coord_equal(xlim=c(-0.05,0.05),ylim=c(-0.05,0.15)) +
theme_minimal() +
geom_point(data=moderns_pl2 %>% filter(Country!="Brazil" & Country!="Mexico"),
aes(x=PC1, y=PC2, col=Country),
alpha=0.5 ## Makes points semi transparent.
+
) geom_point(data=moderns_pl2 %>% filter(Country=="Brazil" | Country=="Mexico"), #Plotting the additional American populations separately to keep colors for other countries the same between the plots
aes(x=PC1, y=PC2),
alpha=0.5 ## Makes points semi transparent.
+
) geom_point(
data=PCA_poplist2_ev %>% filter(Ind=="MA1.SG"), ## Extract MA1 from the entire dataset
aes(x=PC1, y=PC2), ## Set the x and y axes for this set of points
pch=17 ## Change shape of point to solid triangle
)
PCA_plot_2ggsave("scratch/PCA_plot_2.png")
```

And a PCA with all populations, including Mal’ta, used or estimation (no projection)

```
## First we reorient the PCA
<- PCA_all_pops_ev %>% select(Ind, PC1, PC2) %>% filter(Ind %in% c("HGDP00607", "Sir50"))
corner_inds_ap if (corner_inds_ap$PC1[1] > corner_inds_ap$PC1[2]) { PCA_all_pops_ev <- PCA_all_pops_ev %>% mutate(PC1=-PC1)}
if (corner_inds_ap$PC2[1] > corner_inds_ap$PC2[2]) { PCA_all_pops_ev <- PCA_all_pops_ev %>% mutate(PC2=-PC2)}
<- PCA_all_pops_ev #%>% filter(Pop %in% poplist2$Pops) ## Poplist 2 contains all the present-day populations.
moderns_ap
## Then we plot the output
<- ggplot() +
PCA_plot_ap coord_equal(xlim=c(-0.05,0.05),ylim=c(-0.05,0.15)) +
theme_minimal() +
geom_point(data=moderns_ap %>% filter(Country!="Brazil" & Country!="Mexico"),
aes(x=PC1, y=PC2, col=Country),
alpha=0.5 ## Makes points semi transparent.
+
) geom_point(data=moderns_ap %>% filter(Country=="Brazil" | Country=="Mexico"), #Plotting the additional American populations separately to keep colors for other countries the same between the plots
aes(x=PC1, y=PC2),
alpha=0.5 ## Makes points semi transparent.
+
) geom_point(
data=PCA_all_pops_ev %>% filter(Ind=="MA1.SG"), ## Extract MA1 from the entire dataset
aes(x=PC1, y=PC2), ## Set the x and y axes for this set of points
pch=17 ## Change shape of point to solid triangle
)
PCA_plot_apggsave("scratch/PCA_plot_ap.png")
```

### 8.2.4 PLINK MDS

Now let’s get an MDS plot for pairwise distances for the same dataset

```
##Convert package to PLINK format
./trident genoconvert -d scratch/forged_package --outFormat PLINK
ls scratch/forged_package/
# Compute pairwise distances of all individuals
plink --bfile scratch/forged_package/PCA_package_1 --distance-matrix --out scratch/pairwise_distances
```

```
## Read in individual IDs from MDS results
<- readr::read_tsv("scratch/pairwise_distances.mdist.id", col_types="cc", col_names=c("Population", "Poseidon_ID"))
inds inds
```

```
<- janno::read_janno("scratch/forged_package/PCA_package_1.janno", to_janno=F)%>% select(Poseidon_ID, Latitude, Longitude, Country) %>% mutate(Longitude=as.double(Longitude), Latitude=as.double(Latitude))
metadata
## Finally, we add the Lat/Lon information to our datasets
<- left_join(inds, metadata, by="Poseidon_ID")
inds
<- matrix(scan("scratch/pairwise_distances.mdist"), ncol=nrow(inds))
dist_mat dim(dist_mat)
```

` ?heatmap`

```
# first try and filter for a few populations:
unique(inds$Population)
<- inds$Population %in% c('French', 'Greek', 'Nganasan')
indices head(indices, 40)
## Generate a heatmap of pairwise distances
heatmap(dist_mat[indices,indices], labRow = inds$Population[indices], labCol = inds$Population[indices])
```

```
library(ggplot2)
library(magrittr) # This is for the pipe operator %>%
<- cmdscale(dist_mat)
mds_coords colnames(mds_coords) <- c("C1", "C2")
<- tibble::as_tibble(mds_coords) %>%
mds_coords ::bind_cols(inds)
dplyr mds_coords
```

```
<- mds_coords %>% dplyr::select(Poseidon_ID, C1, C2) %>% dplyr::filter(Poseidon_ID %in% c("HGDP00607", "Sir50"))
corner_inds if (corner_inds$C1[1] > corner_inds$C1[2]) { mds_coords <- mds_coords %>% mutate(C1=-C1)}
if (corner_inds$C2[1] > corner_inds$C2[2]) { mds_coords <- mds_coords %>% mutate(C2=-C2)}
ggplot(mds_coords) +
geom_point(data=mds_coords %>% filter(Country!="Brazil" & Country!="Mexico"), aes(x=C1, y=C2, col=Country),
alpha=0.5 ## Makes points semi transparent.
+
) geom_point(data=mds_coords %>% filter(Country=="Brazil" | Country=="Mexico"), #Plotting the additional American populations separately to keep colors for other countries the same between the plots
aes(x=C1, y=C2),
alpha=0.5 ## Makes points semi transparent.
+
) theme_minimal() +
coord_equal() +
geom_point(
data=mds_coords%>% filter(Poseidon_ID=="MA1.SG"), ## Extract MA1 from the entire dataset
aes(x=C1, y=C2), ## Set the x and y axes for this set of points
pch=17 ## Change shape of point to solid triangle
)ggsave("scratch/MDS_plot_ap.png")
```

```
## How does MDS compare to PCA if we restrict to the populations in poplist1?
## Read in the poplist
<- readr::read_tsv("pca_mds_working/PCA_poplists/PCA_poplist1.txt", col_names = "Pops", col_types = 'c')
poplist1
## Filter distance matrix
<- inds$Population %in% poplist1$Pops
indices_pl1
dist_mat[indices_pl1, indices_pl1]
```

```
## Do MDS
<- cmdscale(dist_mat[indices_pl1,indices_pl1])
mds_coords_pl1 colnames(mds_coords_pl1) <- c("C1", "C2")
<- tibble::as_tibble(mds_coords_pl1) %>%
mds_coords_pl1 ::bind_cols(inds %>% dplyr::filter(inds$Population %in% poplist1$Pops))
dplyr
mds_coords_pl1
## Reorient
<- mds_coords_pl1 %>% dplyr::select(Poseidon_ID, C1, C2) %>% dplyr::filter(Poseidon_ID %in% c("HGDP00607", "Sir50"))
corner_inds_mds1 if (corner_inds_mds1$C1[1] > corner_inds_mds1$C1[2]) { mds_coords_pl1 <- mds_coords_pl1 %>% mutate(C1=-C1)}
if (corner_inds_mds1$C2[1] > corner_inds_mds1$C2[2]) { mds_coords_pl1 <- mds_coords_pl1 %>% mutate(C2=-C2)}
## Plot
ggplot(mds_coords_pl1) +
geom_point(data=mds_coords_pl1 %>% filter(Country!="Brazil" & Country!="Mexico"),
aes(x=C1, y=C2, col=Country),
alpha=0.5 ## Makes points semi transparent.
+
) geom_point(data=mds_coords_pl1 %>% filter(Country=="Brazil" | Country=="Mexico"), #Plotting the additional American populations separately to keep colors for other countries the same between the plots
aes(x=C1, y=C2),
alpha=0.5 ## Makes points semi transparent.
+
) theme_minimal() +
coord_equal()+
geom_point(
data=mds_coords%>% filter(Poseidon_ID=="MA1.SG"), ## Extract MA1 from the entire dataset
aes(x=C1, y=C2), ## Set the x and y axes for this set of points
pch=17 ## Change shape of point to solid triangle
)
ggsave("scratch/MDS_poplist1.png")
```

### 8.2.5 Compare plots

Let’s now sum up, by directly comparing all the plots we have generated:

- PCA estimated using modern Eurasian genomic data with Mal’ta projected (“PCA_poplist1”)
- PCA estimated using modern Eurasian and Native American genomic data with Mal’ta projected (“PCA_poplist2”)
- PCA estimated using modern Eurasian and Native American, and Mal’ta genomic data with no projection (“PCA_all_pops”)
- MDS estimated using modern Eurasian, and Mal’ta genomic data (“MDS_plot_ap”)
- MDS estimated using modern Eurasian and Native American, and Mal’ta genomic data (“MDS_poplist1”)

The dataset excluding Native American data comprises much less variation that makes up PC2 and hence the PC2 variation of Eurasians in plot A is stretched up compared to these in plots B and C (including Native Americans). In plot B and C it is therefore more difficult to observe differences within Eurasians along PC2 than in plot A. The variation making PC1 is comparable between the three plots. Individual Mal’ta is more closely related to Native Americans than an average Eurasian, so without Native Americans in the dataset (A) it ends up within the Eurasian variation as there is no Native American genetic signal present that would “pull” him away from the Eurasian variation towards the American variation. When Mal’ta is included in the estimation in plot C (not only projected, like in A and B), we can observe the effects of missing data, inherent for ancient genomes, causing this individual to be pulled towards the plot’s origin.

In MDS the missingness in Mal’ta’s data does not affect its position as it does in PCA. Inclusion of the diverged Native American data in the estimation does cause the decrease of the distances with the Eurasian population along the C2, but the difference between plot E and plot D is not as pronounced as between B and A.

## 8.3 Conclusions

In PCA it is important to estimate the Eigenvalues using high-coverage samples, hence usually modern datasets, such as the 1000 Genomes Project (1kGP) or Human Genome Diversty Project (HGDP), are used for the estimation and the ancient samples are then projected onto the estimated PCs. Also, as mentioned above in the “Shrinkage” section, it is a good approach to project also some modern data if they are to be directly compared to the ancient samples.

While MDS will be less sensitive to the effects of missing data, it disregards the uncertainty of the underlying pairwise distance estimates.

It is thus the best practice to perform both analyses and compare them taking the shortcomings of each into account when interpreting the relative positions of the studied individuals/populations obtained using these methods.