This is a basic introduction to get you started using Skittle. Follow these steps and you should be browsing through genomes in no time.

1. Download

Download the file from the Download page. The executable should work on any modern windows platform. You'll need to extract Skittle.exe and chrY-sample.fa and place them in the folder that you want to store Skittle in for use.  Use the Open file dialog to find the chrY-sample.fa.  Once you've selected it, press ok.

 Skittle will read the whole FASTA file to memory, then display the start of the file.  Your screen should look something like this:

Understanding the Nucleotide Display

The main goal of Skittle is to provide a visual environment in which the user can explore DNA sequences and learn valuable things about that sequence. Your first screen should look like an array of colored squares, referred to here as pixels. Each pixel represents one nucleotide in the FASTA file. One color is assigned to each of the four nucleotides:

G = Green
C = Red
A = Black
T = Blue

N is represented in gray. If your screen comes up a solid block of gray, it's because some sequence files start out with blocks of N's that represent unsequenced regions. Skittle is simply a visual representation of the raw data file so you can read the image like you would a block of text. Starting in the upper left corner, if the pixels are green-black-black-red-blue then the sequence is GAACT. The sequence reads from left to right and then when it reaches a specified width, it returns to the beginning of the next line, just like a type writer. This is what gives GFA it's two dimensional aspect. If this is a large sequence, Skittle only displays a little bit at a time (around 40,000 nucleotides), like looking at the first page in a book.

Program Variables

In the bottom right hand corner you will see a series of five variables: Display Width, Scale, Zoom, Start Position, and Display length.  These are all the essential settings that determine what part of the file you are currently viewing and how it looks.  You can experiment with these numbers to see what each one does:

• Display Width: The all important width is the number of nucleotides in each line of pixels.
• Scale: The number of nucleotides represented by a single pixel.
• Zoom: How large the display appears.
• Start position: the index of the first nucleotide currently displayed (starts in the upper left corner).
• Display Length: Number of nucleotides (not pixels) currently being displayed.

Some of the variables will interact with each other.  For example, doubling the scale also doubles the width and display length.  Display Length is particularly likely to be changed so it does not have the small up/down arrow built in.  Display Length is mainly listed for convenience.  Tools (below) will allow you to change the width and start position in a more natural way.

Basic Tools

Now that we understand the basic image, it's time to start browsing.  Use the buttons on the left side of the screen to select the tool that you want to use.  These will change your mouse pointer to show what tool you are currently using.  The default tool is 'Resize'.  Try clicking and dragging the mouse on the main display. 

Resize:  The Resize tool allows you to change the 'Width' and 'Start Position' settings using the mouse.  Moving from side to side will change the width. Moving the mouse up and down scroll to new starting positions in the FASTA file.  Since the display is laid out in the same way as a scrolling text file 'up' is the start of the file and 'down' is the end of the file.

Move: The Move tool is simply an easy way to use the scroll bars.  It will scroll the screen up and down or side to side.

Select: Use this tool when you want to know the exact position or sequence at a given location.  The cursor is a cross-hair that you can point at a specific pixel.  Clicking a pixel will dump the information to the text box at the bottom of the screen.  The Select tool has a yellow pixel directly underneath the cursor as an aid.  If you are having trouble being exact, try increasing the "Zoom" variable in the lower right.  In the picture above, the Select tool was used to grab the sequence of the large 61-mer repeat at the beginning of chromosome Y.  If you look closely, you can see a yellow pixel at the first Adenosine (black) of the repeat.

Add Bookmark: This button will not modify your cursor.  Instead it will present you with a dialog for adding a bookmark at your current location.  The Start and End values are automatically set to the size of the window currently displayed, but you may change these to any value.  The output format is the GTF genome annotation.  You can also Load GTF annotation through File > Open GTF File.  If you are adding to an annotation you are currently viewing, keep in mind that it will only read the file when you first open it, so you will have to reopen the file to see changes. 

Scrolling - As a convenience, you may also use the keyboard to change the start position and width.  Click on the main canvas to set the keyboard focus then use the following keys:

• Up Arrow = scroll data up 10 lines
• Down Arrow = scroll data down 10 lines
• Left Arrow = Decrease Display Width
• Right Arrow = Increase Display Width

 Clicking on other boxes will set the keyboard focus to that box.  For example, click on the "Zoom" box and then try using the up or down arrow key.

I suggest scrolling around for a while and getting used to the controls. In the sample file of human chrY you should be able to find abundant repetitive sequences. You can see regions of strong nucleotide bias. You should also notice that as a general rule, A and T (blue and black) cluster into long lines. One of the most important things to realize is that the width at which you view a sequence segment greatly affects how it looks. 

Graph Modes

There are four buttons on the right side of the screen.  Each one of these buttons represents a different Graph Mode, which is a visual representation of the raw data in the FASTA file.  Each mode has its own uses and highlights different aspects. A detailed discussion of each mode is covered in the technical paper.  Clicking each of these buttons will toggle the Graph Mode on or off.

1. Alignment Cylinder - 3D cylinder alignment.  Only works at Scale = 1.
2. Nucleotide Display - the simplest display where nucleotides equal colors.
3. Repeat Overview - Color indicates the best alignment from 1-250.  The brighter a color is the better the match.  Areas that don't align well appear as darker colors.  (Minimum Scale = 4)
4. Repeat Map - repeat detection. Similar sequences appear light colored, right-click to align.

Cylinder, Nucleotide, and Repeat Map shown side by side.  The cylinder appears shorter because the three dimensional arrangement is more compact.

Nucleotide Display and Repeat Overview shown at Scale = 20bp per pixel.  The spectrum across the bottom shows which colors correspond to a specific alignment offset.

At Scale = 52 we can see the beginning and end of the chrY-sample.fa file.  After you're done exploring, it's time to go get some new files to study. 

FASTA Downloads:

UCSC Genome Browser Downloads Page

NCBI