Please note: Plots (Histogram, Count and distribution, Pair plot, Box plot, Violin Plot, Joint plot, Scatter plot, Correlation plot) comparing the candidate genes with the other maize genes is generated using a randomly downsampled set of other maize genes. The size of the randomly downsampled genes is equal to the size of the user defined candidate genes.

Example Candidate gene list:

To demonstrate the potential use case of the user candidate gene analysis, we gathered a set of fifty stress genes that were differentially expressed between the control and salt stress samples and used them to identify unique characteristics common among salt stress genes .

Please go through the below explanation for details on table and plot interpretations

Table column interpretation:

For the open chromatin (quantified by ATAC-seq), scores were calculated for three regions: one using the gene body, defined as the region from the annotated transcription start site to the annotated transcription stop site, a second for the upstream region, defined as a 2 KB region directly upstream of the transcription start site, and a third for the downstream region, defined as the 2 KB region directly downstream of the transcription stop site .


Two features were calculated for each genomic interval:

* The proportion of that interval covered by peaks using methods previously described in (Lloyd et al., 2017) :

1. ATAC cov upstream ZmMC

2. ATAC cov genebody_ZmMC

3. ATAC cov downstream ZmMC


* The maximum intensity among peaks overlapping that interval :

4. upstream ATAC intensity ZmMC

5. genebody ATAC intensity ZmMC

6. downstream ATAC intensity ZmMC


7. Labels

The labels are the classes or the groups the genes are mapped into.The labels can act as both target variable or feature as per the need of the user for solving their specific problem

7.1 No Label

This selection is provided to enable users to view the properties of all genes without labeling them into different gene categories or annotations. This is to let users examine the features of multiple genes and identify common patterns among them. As it involves the inspection of all the genes therefore they work only with the "Submit for analysis" button .

7.2 Classical Genes

Classical genes can be defined as the most well studied genes mainly for their visible mutant phenotype (for example: liguleless3).

7.3 Pan-genome Genes

A gene in a given taxonomic group is either present in every individual (core), or absent in at least a single individual (dispensable) and so on .

7.4 Origin Genes

Gene duplication is an important evolutionary mechanism allowing new genetic material and thus opportunities to acquire new gene functions for an organism. There are different origins of duplications such as whole-genome duplication, tandem and duplications, etc.


Graph interpretations:

To the top right corner of the plots/graphs, there are also options to download plot, zoom-out/zoom in, reset axes, autoscale, toggle spike lines, show closest data on hover, compare data on hover, box select,Pan and lasso. Users can also select specific legends to view data only for the selected legends. Details on the interactive plot options are available here:
Interactive graph features

1. Marginal Plot

The Marginal plots are histrogram plot showing the frequency distribution of the selected gene features alonghwith higlighting the candidate genes. This plot will enable the user to easily identify where thier candidate gene lie among the other maize genes for the selected feature.

2. Histogram

The Histogram shows the frequency distribution of the selected ATAC-seq feature such as ATAC cov upstream ZmMC, upstream ATAC intensity ZmMC, downstream ATAC intensity ZmM etc. The X-axis in the histogram represents the range of values present in the selected ATAC-seq. The Y-axis represents the frequency of values. In addition to the graph,to increase the interpretability of the data, We have also included P-values,mean and standard deviations of the selected datasets.

3. Count and distribution

The Count and distribution plot is a smoothed, continuous version of a histogram estimated from the data. The most common form of estimation is known as kernel density estimation.The x-axis is the value of the selected ATAC-seq just like in a histogram and the y-axis in a distribution plot is the probability density function and not a probability. The difference is the probability density is the probability per unit on the x-axis. In general the y-axis on the distribution plot is a value only for relative comparisons between different categories like classical and other genes.

4. Pair plots

The pairs plot builds on two basic figures, the distributions and the scatter plot. The distributions on the diagonal allows us to see the distribution of a single selected ATAC-seq while the scatter plots on the upper and lower triangles show the relationship (or lack thereof) between two variables such as ATAC cov upstream ZmMC and upstream ATAC intensity ZmMC.

5. Box plots

Boxplots are a measure of how well distributed the data in the selected ATAC-seq option. It divides the data set into three quartiles. This graph represents the minimum, maximum, median, first quartile and third quartile in the selected ATAC-seq. It is also useful in comparing the distribution of data across data sets by drawing boxplots for each of them such as the core, non-core, dispensable and private genes. Boxplots can be used to:

6. Violin plots

Violin plots are similar to box plots, except that they also show the probability density of the selected ATAC-seq at different values. These plots include a marker for the median of the data and a box indicating the interquartile range, as in the standard box plots. Overlaid on this box plot is a kernel density estimation. Like box plots, violin plots are used to represent comparison of a variable distribution such as ATAC cov upstream ZmMC, upstream ATAC intensity ZmMC (or sample distribution) across different "categories" (Classical/Other or Core/Non-core) .
A violin plot is more informative than a plain box plot. In fact, while a box plot only shows summary statistics such as mean/median and interquartile ranges, the violin plot shows the full distribution of the selected ATAC-seq.

7. Joint plots

A Jointplot comprises three plots. Out of the three, one plot displays a bivariate graph(scatter plot) which shows how the one variable( such as ATAC cov upstream ZmMC) varies with the another variable(such as upstream ATAC intensity ZmMC). Another plot is placed horizontally at the top of the bivariate graph and it shows the distribution in the form of marks along an axis for each one of the selected ATAC-seq dataset( for example rug plot of ATAC cov upstream ZmMC and upstream ATAC intensity ZmMC). The third plot is placed on the right margin of the bivariate graph with the orientation set to vertical and it shows the distribution of again the two selected ATAC-seq dataset.
It is very helpful to have univariate and bivariate plots together in one figure. This is because the univariate analysis focuses on one variable, it describes, summarizes and shows any patterns in your data and the bivariate analysis explores the relationship between two variables and also describes the strength of their relationship.

8. Scatter plots

A Scatter plot is a great way of exploring relationships or patterns in data. But adding a regression line can make those patterns stand out . Therefore Scatter plot with simple linear regression for the selected ATAC-seq, explains the strength of the relationship between the two variables such as ATAC cov upstream ZmMC or upstream ATAC intensity ZmMC in your scatter-plot using R2, the squared correlation coefficient.It is always between 0 and 1. Higher R2 indicates stronger relationship between the selected ATAC-seq .

9. Correlation plots

Correlation heatmap is graphical representation of correlation matrix representing correlation between different selected ATAC-seq. Correlation ranges from -1 to +1. Values closer to zero means there is no linear trend between the two variables. The closer to 1 the correlation is the more positively correlated they are; that is as one increases so does the other and the closer to 1 the stronger this relationship is. Correlation plots also alerts us to potential multicollinearity problems.

10. Dendrogram plots

A dendrogram is a type of tree diagram showing hierarchical clustering — relationships between similar sets of genes based on the selected ATAC-seq. They are frequently used in biology to show clustering between genes or samples, but they can represent any type of grouped data.
The branches in the dendrogram are arranged according to how similar (or dissimilar) they are. branches that are close to the same height are similar to each other; branches with different heights are dissimilar — the greater the difference in height, the more dissimilarity. Also the different clusters of genes based on the selected ATAC-seq are marked by different colors.

11. Hierarchical Scatterplot

The Hierarchical Scatter plot is a type of pair plot that can be used to visualize the relationship of different pairs of ATAC-seq on the clusters of genes formed during dendrogram.Therefore to create Hierarchical scatterplot we need to input the number of clusters in the choose cluster input box. For the Hierarchical Scatter plot, we need to specify the number of clusters we want to view. Moreover, this number of clusters corresponds to the number of clusters formed in the Dendrogram plot, so we perform this analysis only after studying the Dendrogram plot. The Hierarchical Scatter plot is dynamically sized for the number of clusters, so every user can analyze the dataset based on how many clusters they believe the selected data is forming .

12. Hierarchical Heatmap

Hierarchical clustering heatmap is an intuitive way to visualize information from complex data. It’s also called a false colored image, where data values are transformed to color scale. Heat maps allow us to simultaneously visualize clusters of samples(selected ATAC-seq) in the column and features (genes) in the rows.
First hierarchical clustering is done of both the rows and the columns of the data matrix. The columns/rows of the data matrix are re-ordered according to the hierarchical clustering result, putting similar observations close to each other. The blocks of ‘high’ and ‘low’ values are adjacent in the data matrix. Finally, a color scheme is applied for the visualization and the data matrix is displayed. Visualizing the data matrix in this way can help to find the variables(selected ATAC-seq) that appear to be characteristic for each sample cluster.

13. Heatmap

A heatmap is a plot of rectangular data as a color-encoded matrix.This is a great way to visualize data, because it can show the relation between variables (selected ATAC-seq) including genes. The heatmap here shows the relation between the first 100 genes and the selected ATAC-seq values to reduce time and complexity .

PCA

PCA reduces the high-dimensional interrelated data to low-dimension by linearly transforming the old variable into a new set of uncorrelated variables called principal component (PC) while retaining the most possible variation.
The first component has the largest variance followed by the second component and so on. The first few components retain most of the variation, which is easy to visualize and summarize the features of original high-dimensional datasets in low-dimensional space. PCA helps to assess which original samples are similar and different from each other.
In our case when the selected number of ATAC-seq are higher than 2 then, it is arduous to visualize them at the same time to interpret the genes.PCA transforms them into a new set of variables (PCs) with top PCs having the highest variation. PCs are ordered which means that the first few PCs (generally first 3 PCs but can be more) contribute most of the variance present in the original high-dimensional dataset. These top first 2 or 3 PCs can be plotted easily and summarize the features of all original variables (selected ATAC-seq).

14. PCA 2D variables cluster

A PCA 2D variables cluster plot shows how strongly each characteristic or variable influences a principal component. In the plot we can see these vectors(variables) are pinned at the origin of PCs (PC1 = 0 and PC2 = 0). Their project values on each PC show how much weight they have on that PC.

15. PCA 2D observation cluster

A PCA 2D observation cluster plot showing clusters of samples/observations based on their similarity.PCA does not discard any samples or observations. Instead, it reduces the overwhelming number of dimensions by constructing principal components (PCs). PCs describe variation and account for the varied influences o the original characteristics. Such influences, or loadings, can be traced back from the PCA plot to find out what produces the differences among clusters.
Observations further out are either outliers or naturally extreme observations. Plot observations are annotated with shapes and colors to highlight gene models and gene types for example (Classical/Others). In this way, we can easily hover over any data point, especially outliers, to find gene models with extreme observation values across various sample types. (selected ATAC-seq)

16. PCA 2D biplot cluster

PCA biplot = PCA observations plot + variable plot.
we can probably notice that a PCA biplot simply merges an usual observations PCA plot with a plot of variables. The arrangement is like this:
*Bottom axis: PC1 score.
* Left axis: PC2 score.
In other words, the left and bottom axes are of the PCA observations plot — use them to read PCA scores of the observations (dots).

Another nice thing about these plots: the angles between the vectors/variables tell us how characteristics or variables correlate with one another.
* When two vectors are close, forming a small angle, the two variables they represent are positively correlated.
*If they meet each other at 90°, they are not likely to be correlated.
*When they diverge and form a large angle (close to 180°), they are negatively correlated.

17. PCA 3D variables cluster

This is similar to the PCA 2D variables cluster plot but now instead of two principal components, we can have three PC'S that contribute to most of the variance present in the original high-dimensional dataset. These top 3 PCs can be plotted easily in 3 dimensional space and summarize the features of all original variables (selected ATAC-seq) .

18. PCA 3D observation cluster

This is similar to the PCA 2D observation cluster but now instead of two pricipal compotents, we can have three PC'S that contribute to most of the variance present in the original high-dimensional dataset. These top 3 PCs can be plotted easily in 3 dimensional space and summarize the variance in the observations .
A PCA 3D observation cluster plot displays how much variation each principal components captures from the data. If the first three PCs are sufficient to describe the essence of the data.

For further details on these features please go through the below paper :
https://doi.org/10.1101/730473