Phylogenetic Exercise using Biology Workbench

Outcomes - You learn how to pose questions of phylogenetic relationships, including analysis of viral mutation and adaptation to a host (HIV) or studying evolution and phylogenetic relations of viruses, HIV-SIV-HTLV-STLV, Prions, or SARS. We'll do three different exercises in this assignment:

• HIV mutation study - following 15 HIV seropositive IV drug users at 6 months intervals, looking at viral diversity and divergence, CD4 counts, and progression of the disease in each individual. This is a complex multivariant topic, but easy to explore with Biology Workbench. You might want to familiarize yourself with the BioQUEST PowerPoint presentation about this HIV mutation study.
• Viral envelope evolution study - we will compare the envelope protein in four retroviruses (HIV, SIV, HTLV, and STLV), a key feature for cell attachment, to trace the relationships of retroviruses. We'll apply the same approach to study Prions. A big component of this exercise is using NCBI whole genome resources to efficiently extract and compile sequences for each protein in complex viruses.
• Applying the same techniques to SARS, we'll attempt a comparison of various Coronaviruses to SARS, recreating the early efforts of the CDC and WHO in trying to figure out where SARS came from, and approaches to treat SARS based on existing treatments to Coronaviruses. As above, much of this effort entails finding genomic sequences form NCBI and compiling documents for protein comparisons.
• Most recently, we have added a phylogenetics study to study the evolution of Influenza A.

Plan to spend two to four hours on any of these activities if you are starting from scratch. If you are using pre-built documents of sequences, you can focus on the phylogenetic relationships using Biology Workbench, but you'll miss out on the critical step of hunting down and annotating protein sequences. Try to remember that most of science is collecting and preparing data for more rigorous analysis, including bioinformatics.

If you have not used Biology Workbench, you'll need a quick demonstration of it, or plan to spend some quality time reading a good tutorial.

You'll need to have three or four sets of data for this assignment. These include:

• HIV mutation study - 15 files that contain the nucleotide sequence for HIV-1 env (envelope) proteins from viral clones detected in each individual at six month intervals for the duration of the study. The zipped archive (or folder) will have files that can be opened with Notepad, but may have no file extension, or a .txt file extension. Open them with Notepad to view the contents of each file, which have a FASTA header.
• HIV envelope comparison - this is a single text file which contains eight sequences. Open it in Notepad to view the contents.
• Prion protein comparison - this a collection or Prion precursor proteins from a dozen organisms. You can add to this document as an exercise by searching for Prions in NCBI or Swiss-Prot. This is a great skill if you want to apply this to other protein comparisons across organisms.
• SARS and coronavirus - Approach this with care; as you'll need to be really careful in constructing your data, or start with the corona_sequence_master.txt document. This file contains sequences from the replicase, spike glycoprotein, membrane (or matrix) and nucleocapsid proteins, for several coronaviruses including Avian infectious bronchitis virus (IBV), Bovine coronavirus, (BCoV), Human coronavirus 229E (HCoV-229E), Murine hepatitis virus (MHV), Porcine epidemic diarrhea virus (PEDV), SARS coronavirus (HCoV), Transmissible gastroenteritis virus (TGEV), and more recently, SARS GZ01. There are a half dozen more coronaviruses that have been compared by CDC and WHO, and this is a very active space. Many of the proteins are labeled as "putative" as they have not empirically characterized - instead they are predicted by open reading frames (ORF). This is a very time consuming activity, and requires a lot of patience and organization. You can use the master document, or compilations in the replicase_1A.txt, spike_glycoprotein.txt, membrane_glycoprotein.txt, or nucleocapsid.txt files. Read the three papers The Genome Sequence of SARS Associated Corona Virus and Model of the SARS Corona Virus, and Molecular Evolution of the SARS Virus. Compare your dendrograms with the ones in The Genome Sequence of SARS Associated Corona Virus.
• Influenza and bird flu - This builds off the influenza exercise that some of you have already started. You will need to locate all of your sequence data and format it correctly. You may also use the H1-H15_AlignAA_fasta.txt file, and or the Neuraminidase_master.txt file. This is a very challenging exercise, and you should try to prepare as much as possible by reading through the material at Influenza.htm The Neuramindase sequences are listed in this document with sequences listed in this Excel spreadsheet.

Okay - so now you're ready! Set up your free account at Biology workbench, and follow the directions below. If you can watch a demo, this will be a lot easier. Write down your user name and password, and find out if your instructor has set up a 'class demo account' that you can use as practice.

Log into your Biology Workbench account and follow the steps below. If it doesn't work, back up, and try again.

1. Select "session tools"
2. Click on "new session"
3. Click on "run"
4. Enter the appropriate description for your session (HIV mutation, HIV evolution, Prion comparisons, SARS phylogeny. etc.)
5. Select nucleic tools (for HIV mutation, protein tools for HIV env, Prion, or SARS studies)
6. Select "add new sequences"
7. Click on "run"
8. Click on "browse"
9. Look for the "HIV data by subject" folder
10. Open one of the subjects (subject 1, subject 15, etc)
11. Click on "open"
12. Click on "upload"
13. Click on "save"
14. Repeat this process until you have uploaded all the data
15. Select (using the checkbox) each of the visits for a given subject that you wish to compare
16. Select "ClustalW"
17. In ClustalW, make sure to check the box for "rooted tree" (dendogram)
18. Click on "run"
19. Click on "submit"
20. You'll get a multiple sequence alignment" and a dendogram
21. You can right click on the dendogram to save as an image, or export as a postscript file (.ps)

Your saved dendograms might look like:

• subject_1.ps (postscript)
• subject_1.pdf (Acrobat - you'll need distiller)
• subject_1.bmp (bit mapped image - you'll need Photoshop)
• subject_1.doc (just dropping the image into Word)

Good luck! RDC

rdcormia@earthlink.net

This lesson is copyrighted using an Educational Common License, and may be used freely without restriction for academic purposes.

Copyright © 2007 Robert D. Cormia - January 20, 2007