PLNT4610/PLNT7690 BIOINFORMATICS

Assignment 1 (Sept. 28, 2017)

Comparative Genomics


This assignment is worth 20% of the course grade.  

Due: Tuesday October 10, 2017.


Background 1,2

DNA replication in circular prokaryotic genomes begins at a single origin  of replication, often visualized as being at 12 o'clock on the circle. Two replication forks propagate in opposite directions,  and replication continues until they meet at the terminus, visualized as being at 6 o'clock on the circle.

In bidirectional replication, each replication fork has both a leading and lagging strand. As the two strands f and c are "peeled apart", DNA synthesis on the leading strand proceeds uninterrupted, while on the lagging strand, DNA must be replicated in short stretches, referred to as Okazaki fragments, which are initiated as the DNA duplex opens up.

Figure1

Thus, as shown in Figure 2, we can define two regions of a circular chromosome, arbitrarily designated A and B, as illustrated at right. If the total length of the bacterial chromosome was L, then region A would span coordinates 1 to L/2, and region B would span coordinates (L/2+1) to L.

Referring to Figure 1, in region A the f strand is template for leading strand synthesis, and in region B, the c strand is the template for leading strand synthesis.

Figure 2

It has been observed that the leading strand tends to be enriched for G, relative to C, particularly at the 3rd positions of codons. There are several proposed mechanisms to explain this observation, all resulting from the extra time that the lagging strand remains single-stranded during DNA replication, relative to the leading strand.1,2. The resultant G-C  skew is therefore a good indicator of which strands are the leading and lagging strands in genomic DNA.

Genome viewers such as Artemis are powerful tools for understanding genome organization and evolution. Artemis is designed to superimpose different types of sequence features and graphs onto the genomic map. In these displays, the relationships between sequence characteristics and features become apparent. In this assignment, we will use Artemis to explore the relationship between GC-skew, genome organization, and transcription.

1. Create a symbolic link to the genomes directory and a list of files to use

The directory /home/plants/frist/courses/bioinformatics/as1/genomes contains a number of GenBank files, each for a different prokaryotic genome. If you were to copy these files to your own directory, you would probably exceed your disk quota. Instead, make a symbolic link to the genomes directory, so that you can easily read these files.

cd $HOME/public_html/PLNT4610/as1

ln -s /home/plants/frist/courses/bioinformatics/as1/genomes
go to your as1 directory

create a symbolic link

The genomes directory will behave, for most purposes, as if it was actually present in your as1 directory. For example, if you run artemis in your as1 directory, when you open a file, the genomes directory will appear to be present.

There are two ways in which the symbolic link will behave differently. If you type 'ls -l genomes', you would see the link itself, not the contents of the directory. To see the contents you would have to include the -L option in the ls command ie. 'ls -lL genomes'.  The other point is that the genomes directory and its contents are not world-writable. That means you can't create files within that directory, nor can you delete or modify files in that directory.

Each student will use 10 of the sequences in the genomes directory for use in steps 2 - 4 of this assignment. So that each student may have a unique set of sequences, download randomlists.sh and save it in your as1 directory. Make sure that it is executable: chmod u+rx randomlists.sh.
Next run the script (./randomlists.sh), which will write your list to a file called AS1seqs_1.nam.  These are the sequences you will work with.

2. (4 points) Create a DNAPlotter Map and save feature information for each sequence

a. DNAPlotter Map
For each genome in your list create a DNAPlotter Map. Make sure to include a track for each of the following:

Feel free to use some creativity with colours and other settings that make it easy to visualize features in the map.

Save your maps as PNG files for inclusion in your reports. In DNAPlotter, choose File --> Save as jpeg/png Image. Use the ".png" extension to distinguish the plot file from the GenBank file. For example, if the Genbank file was Corynebacterium_ulcerans0102.gen, then your map should be saved as Corynebacterium_ulcerans0102.png.

After producing your first map, export your tracks, as described in the DNAPlotter tutorial, to a file whose name includes ".tracks" as an extension. You can import this file to speed up production of your other genome maps.

b. Feature Information
For each genome, save the feature information by going to the feature window in Artemis, right clicking on the mouse, and choosing Save List to File. Use the ".fea" extension to distinguish the plot file from the GenBank file. For example, if the Genbank file was Corynebacterium_ulcerans0102.gen, then your map should be saved as Corynebacterium_ulcerans0102.fea.

3.  (5 points) Modify the existing fea2tsv.sh script to read a feature list from Artemis, and convert it into a Tab-Separated Value (TSV) file.

We can automate procedures using Unix commands by writing those commands in a file referred to as a script. A script implementing steps 1 -5 from the tutorial Extracting features from text files can be found in the file fea2tsv.sh. In this exercise, we'll modify the script with some improvements on the original protocol. 

The problem is as follows: In the Background section above, we have oversimplified things by assuming that for all prokaryotic genomes, the replication origin will always be annotated as starting at position 1. However, the choice of which nucleotide in a circular genome gets specified as position 1 is often an arbitrary location for many genome projects. Consequently, many prokaryotic genomes place the replication at a different position. For this reason, it would be far easier to calculate the f and c values if our TSV file contains annotation for both CDS and rep_origin features.

Fortunately, the grep command can read a file containing a list of patterns, each on a separate line. The output from grep will be any lines from that match any of the patterns. For example, you could create a pattern file called fealist.txt containing the following lines:

^CDS
^rep_origin

 If we typed

grep -f fealist.txt < Corynebacterium_ulcerans0102.fea

any lines beginning with either CDS or rep_origin would be printed to the standard output. (In regular expressions, '^' indicates the beginning of a line. If '^' was not included in the expression, the search would match any line that included CDS or rep_origin anywhere within a line.)

To get started,  save this file in your as1 directory. You will need to make this file executable in order to run the script:

chmod u+x fea2tsv.sh

Before changing anything, try running the unmodified script yourself using one of the feature files generated using Artemis. For example, if your feature file is Corynebacterium_ulcerans0102.fea, you can run the script by typing
./fea2tsv.sh Corynebacterium_ulcerans0102.fea By default, the current working directory is not in your $PATH. Therefore, when we run a script in the current directory, we have to precede its name with ./ to tell the shell that the script is in the current directory.

In this example, output would be written to a file called Corynebacterium_ulcerans0102.fea.tsv.

Your job is to modify the script as follows:

The script is heavily annotated with comment lines ie. lines beginning with a hash mark (#), that explain what each section of code is doing. Places where you need to make changes have comments in ALL CAPS to tell you what changes need to be made.

When the script is complete, you should be able to run it as in the example below:

command
output file name
./fea2tsv.sh  Corynebacterium_ulcerans0102.fea  fealist.txt
Corynebacterium_ulcerans0102.fea.tsv

If you implement both modifications above, your script should be able to read ANY file containing feature lines, not just fealist.txt.

Your TSV file should directly readable by LibreOffice Calc without any modification. Links to some useful tutorials for writing bash shell scripts can be found in the References 3,4,5 below.

Before proceeding to the next, step, use your fea2tsv.sh script to generate a .tsv file from each of your .fea files.

4. (5 points) Quantify transcriptional strand biases for coding sequences.

For each genome, the goal is to quantify the transcriptional bias, that is, the tendency for coding sequences to be transcribed on either the forward or reverse strands, for each of the two regions, A and B, as illustrated in Figure 2 above.

a. Decide on a cut off row that delineates the junction between regions A and B

You can find the length of the genome on the LOCUS line of the GenBank file for each genome. This information is also found in Artemis, using View --> Overview. The replication terminus can be assumed to be at the half way point on the circle, opposite the replication origin. That is, if the origin is position 1, and the sequence is length L, then the terminus would be at position L/2. For example, if the sequence was 2,500,000 bases long, the terminus would be at position 1,250,000.

If the replication origin was at a position other than 1, you would have to calculate the terminus based on that position.
Given the halfway point H = L/2, the location of the terminus T is

if R > H :
    T = R - H
else:
    T = R + H


Next, scroll down the rows of your spreadsheet to roughly the halfway point. Look for a coding sequence whose coordinates overlap the terminus. This row would be the last row in region A. The next row would be the starting point for region B. For example, if there were 4000 CDS sequences total, region A might span rows 1 through 1987 in the spreadsheet, and region B would span rows 1988 through 4000.

b. Calculate the transcriptional bias for regions A and B.

The transcriptional strand bias (TSB) is the degree to which the direction of transcription is skewed either to the forward strand or the reverse strand. It could be calculated as a ratio of the difference between numbers of CDS features on the forward and reverse strands to the total number of coding sequences in each region. That is,

TSB = (f-c)/(f+c)

where

Suppose you had the following data in your spreadsheet:


A
B
C
D
1
CDS
2940
4022
f
2
CDS 4062
4319
f
3
CDS 4347
6278
f
4
CDS 6809
7000
f
5
CDS 7198
8268
f
6
rep_origin
8330
8350
-
7
CDS
8486
8746
c
8
CDS
8855
8980
c
9
CDS
9098
9473
c
10
CDS
9599
10172
f
11
CDS
10279
10546
c

If the CDS sequences in region A spanned the first 5 rows, and those in region B spanned the last 5 rows, you could calculate the TSB values for each region.

For example, to calculate TSB for region A in LibreOffice Calc, you could count the number of CDS features on the forward strand using the formula =COUNTIF(D1:D5,"f"). Similarly the number of CDS features on the complementary strand would be counted using =COUNTIF(D1:D5,"c"). Region B would be calculated similarly. The results would be shown in a table for each species.

region


(f-c)/(f+c)
A
f(1:5) = 5
c(1:5) = 0
 1.0
B
f(7:10) = 1
c(7:10) = 4
-0.6

Save your spreadsheet in LibreOffice Calc format, by choosing File --> Save As. In the Save window, choose "ODF Spreadsheet (.ods) as the format, and Save. For example, if the file you read into the spreadsheet was Corynebacterium_ulcerans0102.fea.CDS.tsv, the file would be exported to Corynebacterium_ulcerans0102.fea.CDS.ods.

5. (3 points) Conclusions - What have you learned?

6. (3 points) Presentation.

Part of the grade will be determined by the quality of your web page(s) for the assignment, including:


How to get started

1. Create a directory called either public_html/PLNT4610/as1 or public_html/PLNT7690/as1. Make this directory world-readable and world executable. (That's as1 - using the number 1, rather than the letter l).

2. Do  all work in the as1 directory. That way, all your files will already be where they need to be.

What you need to complete your assignment

Your report should include the following:

1. Links to your tracks file and your fea2tsv.sh script, and a link to your AS1seqs_1.nam file.

2. For each genome, present your results in a 2-column table, as shown in the sample file. You are expected to follow the file naming conventions used above.

3. A discussion of the main findings from your data. The questions in part 5 above are a starting point, but feel free to add additional observations, explanations, or to state hypotheses arising from the observations. Feel free to make tables, charts, graphs, or anything that will get your points across.
How to post it (3 points)

1. Create a new HTML file called as1/as1.html. Your web page for Assignment 1 should take the form of a report, that makes it easy to figure out what you did.

2. Make all files in the as1 directory world-readable. (chmod a+r  *)

3. Edit either PLNT4610.html or PLNT7690.html to include a link to as1/as1.html.

4. In the Firefox or SeqMonkey Browser, go to your home page and follow all hypertext links to your assignment, and test all links to your output files.

On the day the assignments are due, I should be able to just go to each person's web site and find the output. You don't need to send me an email message saying that your assignment is complete. If you choose not to hand in this assignment, you don't need to do anything.

Academic integrity:
1.Your work is assumed to be your own original work. All University policies regarding academic integrity apply.
2. Show your work. For example, a spreadsheet that had the final answer typed in, rather than calculated by a formula, would not provide any evidence that you had actually done the work.


References
1. Francino MP, Ochman H (1997) Strand asymmetries in DNA evolution. Trends in Genetics 13:240-245.

2. Mclean MJ, Wolfe KH, Devine KM (1998) Base Composition Skews, Replication Orientation, and Gene Orientation in 12 Prokaryotic Genomes.  J. Mol. Evol. 47:691-696.

3. BASH Programming - Introduction HOW-TO [http://tldp.org/HOWTO/Bash-Prog-Intro-HOWTO.html]

4. Chadwick, R (2015) Bash Scripting Tutorial - Ryan's Tutorials [http://ryanstutorials.net/bash-scripting-tutorial/]

5. Gite, V.  (2015) Learning bash scripting for beginners [http://www.cyberciti.biz/open-source/learning-bash-scripting-for-beginners/]