DNA Biology and Bioinformatics Campdnacamp.cgrb.oregonstate.edu/PhylogenyCommands.docx · Web viewto generate a multiple sequence alignment. 8. Once the sequences are aligned, click

************************************************************************STEM CAMP 2015 - DNA Biology and Bioinformatics - Thursday July 16************************************************************************

What we're doing today: Phylogeny and Blast to analyze the evolution of bacterial symbionts from our worms.

From all of your nematodes collected Monday/Tuesday, we performed DNA isolation, PCR and sequencing for two genes: 18S ribosomal RNA (nematode) and 16S ribosomal RNA (Wolbachia endosymbiont).

Part 1: For all the nematodes you collected on Monday/Tuesday, analyze what kind of nematodes they were by looking at the 18S sequences compared with the NCBI GenBank databases.

Part 2: For any of your nematodes with successful 16S sequences, these may be discoveries of new Wolbachia endosymbionts. We will analyse this by aligning them with other Wolbachias and creating a phylogenetic tree.

Files you will use:

1. Your nematode 18S sequences.2. Your bacterial 16S sequences (Wolbachia).3. An alignment file of Wolbachia 16S sequences including other bacteria.

Let's begin!

You will use unix. So, click on PuTTY icon and for "Host name" type:

crick.cgrb.oregonstate.edu

For "Port" type: 732

Click Open.

At the Security Alert, click Yes.

Once the terminal (black window opens), at "login as:" type YourName_wsand then type your password.

Now make a directory for today's work:

>mkdir YourName_Phylo

>cd YourName_Phylo

Copy the files you will need from our teaching folder to your directory:

>cp /nfs1/Teaching/CGRB/rnaseq_analysis_spring_2015/*sequence.fasta .>cp /nfs1/Teaching/CGRB/rnaseq_analysis_spring_2015/*aligned.fasta .

Use Filezilla to do this by sescure ftp. Open Filezilla from the desktop, and for "Host", type:

sftp://crick.cgrb.oregonstate.edu

for "Username", type: Yourlogin_ws , Then enter your Password and for"Port" enter 732. Then hit "Quickconnect".

In Filezilla, drag all the "...sequence.fasta" and "...aligned.fasta" files to your Desktop folder.

Part 1: Exploring your 18S (worm) sequences by online BLAST:

1. Open Internet Explorer or other browser and go to NCBI Blast

http://blast.ncbi.nlm.nih.gov/Blast.cgi

2. Choose "Nucleotide Blast".

3. Choose "Browse" below the main query window and locate your 18S sequence (on Desktop -> Computer# -> Desktop). Under "Program Selection" at the bottom, choose "Somewhat similar sequences". Click "BLAST".

4. After a few minutes, results will appear. Scroll down and look at the list of organisms and genes that result. Notice the % similarity and other values.

You can also look just above "Graphic Summary" by "Other Reports" and click on "Distance tree of results" to see a crude phylogeny.

Questions: Did we sequence the right gene? Did it come from the right organism (a nematode)?

We will talk about everyone's results at this point.

Part 2: Exploring your bacterial 16S by alignment and phylogeny in MEGA.

1. On the lower left on your desktop, from "START" choose "All Programs" then choose "bioinformatics" then choose "MEGA6", and click on the MEGA6 program.

2. In MEGA6, choose "File" then "Open A File/Session" and browse to Desktop -> Computer# -> Desktop to choose Wolbachia_16Saligned.fasta. Under "How would you like to open? choose "Align" mode.

3. Take a look at the sequences. These have names, and colored nucleotides. They are "aligned" by having dashes for gaps.

4. Now add your own 16S sequences by chosing the little icon on the top with the right-facing red arrow "Insert sequences from MEGA/FASTA...". Navigate to Desktop -> Computer# -> Desktop and at the bottom of "Files of type" choose FASTA. Now you will see your 16S sequence. Open it.

5. You will see it is not aligned with the others. Click on Align > Edit/Build alignment and then Retrieve sequences from File and OK. Navigate

6. Click on the Muscle tool to align the sequences using MUSCLE (Align DNA, not codons, and click OK when MEGA asks to select all)

7. Click Compute to generate a multiple sequence alignment.

8. Once the sequences are aligned, click Data then select Phylogenetic analysis from the dropdown menu. Select NO for proteincoding nucleotide sequence data. Click on the original MEGA window. You should see a "TA" and Close Data button indicating that the data has been opened to phylogenetic analysis.

Next, click Phylogeny, Construct/Test Maximum Likelihood Tree, and Yes to use the currently active data. Change the "Test of Phylogeny" from "None" to "Bootstrap method" and change 500 to 50. Change Model/Method to Kimura 2-parameter model and change Rates among sites to Gamma.

What does the phylogeny show for the orgins of the Wolbachia endosymbiont in nematodes?

We will talk about everyone's results at this point.

Example: From the figure below, how many orgins of the "Meckelian Groove" probably occurred in evolution?