Identification of Unknown Viruses

Part A: Virus Identification by Microarray Hybridization

In this exercise, you will be assigned a sample containing an unknown virus, and you will use microarray hybridization [1, 2] to identify the virus.

1. Use the microarray hybridization simulator to hybridize your unknown to the virusID microarray.
2. Examine the pattern of spots to identify the virus. Here are several possible approaches:
   • Identify some individual bright spots, and look up which virus(es) those spots are designed to detect.
     • Select a particular bright spot on your microarray image.
     • Determine the number of the spot. Spots are numbered right to left, top to bottom within each zone. There are 10 rows and 10 columns (one hundred spots) per zone, and ten zones (one thousand spots) per metarow.
     • Look up the spot number in the array design Excel file (~1 MB). (The full design file, including reporter sequences, is 25 MB.
     • Repeat for several more spots, to see if you get a consistent answer.
   • Look through the virus catalog to compare the hybridization patterns of known viruses to your unknown.
   • Use a machine learning algorithm to classify your sample. This is best done using numbers obtained with feature extraction software, such as Scanalyze.
     • Use clustering on your sample's measurements together with known samples, and see if it clusters tightly with any of the known values.
     • Use a Bayesian Classifier.
     • Try some other approach (neural network, decision tree, etc.).

Part B: PCR/Sequencing of Virus Unknowns

PCR amplification using virus specific primers will assist in further classifying your viral unknown. These instructions assume you have already used the VirusID microarray to determine the general category of your unknown virus.

1. Open the web page for the PCR simulation, located at http://www.cybertory.org/cgi-bin/pcr/pcrsetup.cgi. Enter your user name; you can make up your own user name, but be sure no one else uses the same name! Use the default values of 8 reactions, with 2 primers per reaction. The PCR setup page lets you choose parameters for each individual reaction. To speed up the process, you can make choices in the "Default Reaction Parameters" form; these choices will be copied into all reactions.

2. A link to a page containing primer sequences which selectively amplify various respiratory viruses is located on the top left part of the PCR simulator web page. Copy and paste the forward and reverse PCR primers you wish to test against your unknown into the simulator.

3. Select your virus unknown in the templates pull down menu. When all reactions are set up, click "run reactions and show gel" at the bottom of the PCR setup page.

4. Your eight PCR reactions will be run on a simulated gel. By modifying the cycling conditions, you can affect the specificity of the PCR reaction. You can observe the effects of increasing cycle number and primer concentration on the formation of the PCR product, although in this exercise there is no human genomic DNA to interfere with your reaction at low temperature of priming.

5. It is advisable to run all of the available primers from the various publications. The Templeton primers are the most "bullet proof" primers, but the amplification products are small, and the region of sequence they amplify is highly conserved (and thus not very informative in terms of telling subtypes apart).

6. To sequence your PCR product, click on the link to the trace generator at the top right hand side of the PCR simulator webpage.

7. Enter your name to start the trace generator (sequence simulator).

8. The trace generator will automatically have the last set of PCR reactions you have run listed in the template pull-down menu.

9. Choose the forward PCR primer as the sequencing primer.

10. Run the trace generator. You can modify the sequencing conditions and observe the effect on the sequence quality.

11. When the simulator is done, save your sequence.

12. Open the Trev program (trace viewer from the Staden package of sequence analysis software).

13. In the File pulldown menu, select Open (your .scf file from the trace generator). Make sure that .scf files are selected as the file type.

14. You should see the trace file (as a four-color florescent electropherogram). Evaluate the quality of the sequence.

15. If the sequence is of sufficient good quality, save the file as a text file. Select the File pulldown menu, the save option allows you to save as a text file.

16. Save it as a .txt file and open it in wordpad.

17. Convert the text file to a linear string of characters (remove all spaces and additional text). Go to the Sequence Massager webpage at http://www.attotron.com/cybertory/analysis/seqMassager.htm

18. Copy and paste the .txt file contents into the box and select "remove FASTA comments", "remove line breaks" and "remove white spaces".

19. Go to the LANL influenza database or the NCBI website to BLAST the sequence to further identify the species of virus. Copy and paste the ".txt" file into the BLAST function of either website (http://www.flu.lanl.gov/, or http://www.ncbi.nlm.nih.gov/).

References

1. Wang D, Coscoy L, Zylberberg M, Avila PC, Boushey HA, et al. (2002) Microarray-based detection and genotyping of viral pathogens. Proc Natl Acad Sci USA 99: 15687-15692. PubMed

2. Wang D, Urisman A, Liu YT, Springer M, Ksiazek TG, Erdman DD, Mardis ER, Hickenbotham M, Magrini V, Eldred J, Latreille JP, Wilson RK, Ganem D, DeRisi JL. Viral discovery and sequence recovery using DNA microarrays. PLoS Biol. 2003 Nov;1(2):E2. Epub 2003 Nov 17. PubMed