TUTORIAL:

DISPLAYING AND MANIPULATING SEQUENCES - BioLegato

Sept. 23, 2021

NUMSEQ documentation: $doc/fsap/numseq.txt
BACHREST documentation: $doc/fsap/rest.txt

If you have already done the previous tutorial on "Displaying and manipulating sequences at the command line", you can skip step 1, since you'll already have these sequences in our sequence directory.

1. Copy sample sequences to your $HOME/sequence directory

{brassica:/home/plants/frist}cd                 do next step if $HOME/tutorials doesn't exist
{brassica:/home/plants/frist}mkdir tutorials     create directory for this tutorial
{brassica:/home/plants/frist}mkdir tutorials/sequence
{brassica:/home/plants/frist}cd $birch/tutorials/bioLegato/sequence
the location of birch ($birch) is /home/psgendb in this example
{brassica:/home/psgendb/tutorials/bioLegato/sequence}cp *.gen $HOME/tutorials/sequence
copying GenBank files to new directory
return to $HOME directory verify that new files and directories are present
{brassica:/home/plants/psgendb/tutorials/bioLegato/sequence}cd
{brassica:/home/plants/frist}ls -l
drwx------   1 frist    drr     512 Oct 31 10:11 tutorials/
{brassica:/home/plants/frist}cd tutorials
{brassica:/home/plants/frist/tutorials}ls -l
drwx------   3 frist    drr     512 Oct 31 10:11 sequence/
{brassica:/home/plants/frist/tutorials}cd sequence
{brassica:/home/plants/frist/tutorials/sequence}ls -l
-rw-------   1 frist frist     5404 Oct 31 10:13 X52331.gen
-rw-------   1 frist frist    10739 Oct 31 10:13 PBI101TD.gen
-rw-------   1 frist frist     8278 Oct 31 10:13 pBSGUS.gen
-rw-------   1 frist frist     3674 Oct 31 10:13 PEACAB15.gen

Files with the .gen extension are in GenBank format. Since these are ASCII text files, you can view them in any text editor. Double clicking on a file in the file manager will bring up the file in the default text editor for your bioLegato installation.

2. Running NUMSEQ from bldna

bioLegato is a program that runs other programs. bldna is an instance of bioLegato that contains functions and programs specifically for DNA sequences. bldna can be launched either from the BIRCH launcher or from the command line.

As you will see, the program runs in the window in which a bioLegato instance was started. The bioLegato instance generates the keystrokes that you would normally be typing.

To illustrate the point, let's try running NUMSEQ from bldna. We will demonstrate to launch bldna from the command line and from BIRCH.

To launch bldna from the command line.

{brassica:/home/plants/frist/tutorials/sequence}bldna

To launch bldna from BIRCH, locate

From the interface, select the option Sequence --> bldna - DNA sequence analysis system.

This will open another window for bldna.

IMPORTANT NOTES:
1.While a bioLegato instance is running, the terminal window can not be used for other commands. If you need to type commands, open another terminal window. Alternatively, you can launch bldna (or any program) in the background by including the ampersand character at the end of the command eg.
bldna & . The window would now be free to use for other commands.
2. Although bioLegato can read files from any directory, it's best to launch a bioLegato instance from the directory in which you plan to work.

Read in PEACAB15.gen:

File --> Open

Click on the filename, and click 'Open'.

Hint: There are 2 steps to running a program from a bioLegato instance
1. Select sequence(s) - either:

click on a single sequence

To select a group of adjacent names
- click on topmost name
- hold down SHIFT key
- click on bottommost name

To select several sequences that are not adjacent
- hold down the Ctrl key and click each sequences

To select all sequences, choose Edit --> Select All

2. Choose a program from one of the menus

To run numseq, click on PEACAB15 and choose DNARNA --> NUMSEQ.

The numseq menu appears, containing menu items for all parameters in the NUMSEQ Parameters menu.

HINTS ON bioLegato MENUS:

for sliders, you can either choose the number with the slider knob, increment or decrement by clicking on the slider cable, or increment or decrement using the up and down arrow buttons.
always remember to select a sequence before going to a menu. The bioLegato instance has to be told which sequence(s) to work with.

Output goes to a temporary file, and appears in a text editor window

When you click OK, bldna saves the specified sequence in a temporary file, and runs numseq. Numseq reads in the temproary sequence file and prints it out according to the parameters sent to it by bldna. Output is stored in a temporary file, which is opened in a text editor.

Normally, the temporary output file (eg. bioLegato2155281324855234117.tmp.out) will be deleted when you quit the Text Editor window. To save the file, choose File --> Save As and type in a name for the output. It's a good idea to include a .numseq file extension to indicate that this file is output from numseq.

Because the output is ASCII text, you can do lots of things with it, including importing it into a word processor, pasting it into another window, mailing it, or even using it as input for other sequence programs. In the latter case, the output will probably need to be modified to conform to the desired input file format eg. Pearson/Fasta.

Examples:

To view both strands:

set "Both strands"

To translate in 3 reading frames:

TRANSLATION: Yes
Reading frames: Three
write in GROUPs of: 15
number of GROUPs printed per line:

NUMSEQ breaks up the sequence into groups of nucleotides, numbering each group. For translation, GROUP must be divisible by 3, because translation is done in discrete codons of 3 bases each. Because the resultant output line will be 15 groups of nucleotides times 7 groups per line (ie. 105 characters per line), you will have to stretch the output window to prevent text wrapping.

To limit printing to only part of the sequence eg. bases 200 - 400:

START: 200
FINISH: 400
TRANSLATION: No

To view the opposite strand of the same region:

Which strand: opposite
START: 400
FINISH: 200

This example illustrates that creating an opposite strand requires two steps. First, we have to specify the strand as 'o' (opposite) rather than 'i' (input strand). This causes the bases to be complemented. However, if all we do is complement the input strand, then the opposite strand would be printed 3' to 5', because we would be starting at 200 and ending at 400. Therefore, START must be set to 400, and FINISH to 200.

2. Working with circular DNA molecules

Circular DNA molecules require a bit of thought. Since printing is always done 5' --> 3', the direction (clockwise vs. counterclockwise) determines the strand, or vice versa. Consider the Bluescript cloning vector (GenBank X52331). Conceptually, one base must be arbitrarily labeled as 1. In the GenBank entry, 1 is the first base in the file, and 2958 is the last base in the file. In the physical plasmid, of course, base 2958 is adjacent to 1.

In NUMSEQ, the START, FINISH and WHICH parameters govern which parts of the sequence are displayed.

To view the top strand of the PvuI (CGAT^CG) fragment going clockwise from 2417 to 503:

1) START: 2417
2) FINISH: 503
7) WHICH: Original

Since you're only considering 1 strand at a time, you want to start with 2417, which is the 5' end of the small PvuI fragment, on the original strand.

To print the same sequence on the other strand, we can't just change WHICH to 'Opposite".

1) START: 2417
2) FINISH: 503
7) WHICH: Opposite

Try it and you'll see that what you get is the large PvuI fragment going from 2417 to 503, and that this fragment doesn't even terminate where PvuI would cut. It's best to visualize the fragment ends as illustrated below:

So the correct way to print the opposite strand of the small fragment would be:

1) START: 501
2) FINISH: 2415
7) WHICH: Opposite

Example: Simulated restriction digest of a pBluescriptKSm13+ at BamH1
The BamHI site is at 690 on the input strand, meaning that the 5' end of the BamH1 site on the original strand is at position 690. Thus:

1) START: 690
2) FINISH: 689
7) WHICH: Original

If we wanted the inverse complement (ie. counter clockwise), the NUMSEQ parameters would be

1) START: 693
2) FINISH: 694
7) WHICH: Opposite