Tutorial: A Phylogenetic Tree with MEGA 5

Last Friday (September 27, 2013) instead of having an internship, I read the tutorial book my mentor gave me and made my first phylogenetic tree!

First of all, I had to download a program called MEGA 5, which allows us to align and compare related sequences with our sequence of interest. For this tutorial, we use a sequence of an alpha-glucuronidase gene from the bacterium Thermotoga petrophilia. We can obtained the sequence by clicking Do BLAST Search from the Align menu, and it will lead us to the following window:

MEGA 5 is linked to BLAST.

I entered the "accession numbers" and the "query subrange", which are given by the book, chose "Neucleotide collection" for the Database, and blastn for Program Selection.

The results showed up few seconds after I hit search. It gave all the sequences that produce similar alignment to our query sequence. The first entry was eventually my query sequence (because that's what I searched for!) Each entry consists of 8 elements: accession number, description, Max score, Total score, query coverage, E-value, and Max identity. For the sake of time, I will just briefly explained Max score, Total score, query coverage, and E-value. Based on the definitions given by the book,

• Max score - the score for the highest scoring segments of the subject sequence
• Total score - the sum of the scores for all the segments that aligned (including noncontiguous alignments)
• Query coverage (%) - shows how much of the query sequence aligns with the subject sequence.
• E-value - describes the number of hits with scores this high that one would "expect" to see by chance when searching a database of a particular size. The closer the E-value to 0 the better.

 The book asked us to include those subject sequences that have E-value <10^-3 and query coverage >60%. For that, I chose 9 sequences, and I clicked "Add to Alignment" for each (some sequenced require to be reversed before added to alignment).

Sequences of the nine samples

Then I clicked "Align DNA" and get:

This is the end portion of the sequences after being aligned

Finally, I construct a phylogenetic tree from my DNA alignment by choosing "Construct / Test Neighbor Joining Tree" and tada~

My first phylogenetic tree!

This Friday, I will discuss with Dr. Miller more about the assumptions of making phylogenetic tree and do more practices with MEGA 5. I can't wait!:)

Cladisticules

(September 20, 2013) I began my internship today!!! This year I am very lucky to be able to do intern on campus, which cut off all the travelling time:) Dr. Miller, who is my mentor, currently works in UCSD as a part of the CIPRES project (http://www.phylo.org/). He has been developing software and information technologies to assist biomedical research and now works with a supercomputer located in San Diego Supercomputer Center that maintains a database for phylogeneticic tree inference.

Last year, I wrote down for one of the reasons to apply for internship was to "widen my horizon," and here is the opportunity! All of my past experiences have more to do with molecular biology, such as growing cells and running PCRs in a lab. However, this year I get to learn biology in an evolutionary perspective. Dr. Miller raised a good point that studying phylogenetic trees is very critical for it allows us to take a step back and look at the organisms in the most fundamental view, to see a bigger picture of the biological world. I also learned many applications of phylogenetic trees, including predicting the properties of new pathogens, fighting against diseases that destroy food sources, and maintaining biodiversity. Nonetheless, I actually had had some experiences with some database such as BLAST and CLUSTAL before, which would help me through this course.

Different trees have different patterns, which I think are pretty impressive and very beautiful.
http://explorebio.wikispaces.com/The+Art+of+Phylogeny

After the introduction, we began a short exercise - to establish a simple cladogram.

1. There are 8 different species. We described each using different characters. The variants within a character is called character states. For example, the color of the abdomen is a character where as white / black abdomen are the character states.
   
2. Then we made a data matrix, with 0 meaning primary states and 1 being the developed states.
   
3. Based on the matrix, we constructed a Venn diagram.
   
4. Finally, we drew a cladogram based on the Venn diagram.
   

This exercise was actually really tricky because black abdomen rise up in two different places, which is called a homoplastic character. Although I had some basic background from AP Biology, this exercise was harder than expected. Luckily, we now have technologies that help us do this (which is what I will be learning!), and this was just to give me a taste of how long scientists used to spend on sorting out the evolutionary tree.

I definitely learned a lot, including many vocabs, during today's meeting. While Dr. Miller won't be here next week, he gave me a book Phylogenetic Trees Made Easy: A How-To Manual to read. My assignment is to learn to establish a phylogenetic trees on MEGA. Today was a great start and I am ready to explore the beauty of a phylogenetic tree!

Here is the link for the video Dr. Miller showed me during the introduction, and I thought it was pretty cool and well explained: http://archive.peabody.yale.edu/exhibits/treeoflife/film_discovering.html

A Brand New Year - STEM Intern 2013-2014!

September 8th, 2013 Time went by so fast and it has already been a week since the school start! This year, I am really glad that I got the opportunity to participate in EWS STEM Internship program again. However, it feels different doing an internship this year, especially after the intern experiences from last year and from this past summer. Last year, I focused more on learning lab techniques and the principles under systematic and molecular biology, and this past summer I practiced a lot of these techniques such as PCR, digestion, and gel electrophoresis. Thus, this year I hope I can learn more advanced techniques such as western blot.  If possible, I wish to have my own project to work on, which would allow me to be familiar with the process - question, hypothesis, experiments, and results - that a lab project is involved in. Last but not least, I would like to explore my topic of interest (molecular / cellular biology, genetics, oncology) even more in depth. I am very exciting for the program to start, and I believe this year will be another wonderful year!

The image on the left shows a cell undergoing extocytosis, and the image on the right shows the vesicles on dock located in the synapse in the cerebellar cortex. My work for this past summer involved in a protein that participated in intracellular trafficking in yeast. The image is retrieved from Collins Lab at Cornell Univeristy. http://blogs.cornell.edu/collinslab/