Camille B. Troup, Ph.D

Biotechnology Consultant

camitroup@comcast.net

Mr. B is a 42 year old man who has been undergoing treatment for HIV infection for the past two years. He has had difficulty adhering to a multidrug cocktail of HIV protease and reverse transcriptase inhibitors for treatment of his infection. At a recent doctor's visit, he had his blood tested and the doctor reported that his T4 cell count had dropped significantly and his viral load had become elevated since his last visit.

Mr B then began to get more serious about taking his medicine. Unfortunately, at his next doctor's visit, his T4 count was still low and his viral load continued to be high. The doctor then recommended an HIV genotyping test to see if he had developed drug resistance.

The goal of the laboratory is to answer the following questions:

1. Based on the results of the genotyping test, which mutations are present in the virus infecting Mr. B?
2. Based on the Stanford HIV database report, which drugs had Mr. B become resistant to?

The HIV genotyping powerpoint presentation should be viewed prior to attempting the exercise as it contains relevant scientific background for performing the exercise. It can be given as a lecture to the class in approximately 30 minutes. The PowerPoint presentation is freely available online on the Cybertory^TM website at http://www.cybertory.org/exercises. Scroll down to the HIV Genotyping section (second paragraph) and double-click on the "PowerPoint Presentation" link.

The Staden package is sequence analysis software which allows the user to view, process and assemble sequence the chromatograms generated by the Cybertory^TM trace generator. This software package must be downloaded and installed prior to analyzing sequence data generated by the Cybertory^TM trace generator. To install the Staden package, follow the instructions:

1. Go to the Staden website at http://staden.sourceforge.net/ and click on the Downloads link.
2. Under the Staden 1.5.3 section, choose the platform (e.g. staden-macosx-1-5-3.tar.gz for an apple computer or staden-windows-1.5.3.msi for a PC) and select it with the mouse. A page will appear with locations to download the software.
3. Choose one of the download locations and double click to download the Staden software installer.
4. Once the file has been downloaded, simply double click on the installer and follow the installation instructions.
5. The Staden Package will install as a folder of applications and a tutorial in the folder where the Staden installer has been saved.

To begin the exercise, open up three web browser pages and follow the instructions:

1. Set the first web browser page URL to http://www.cybertory.org/exercises. At the middle of the page is the HIV genotyping exercise section. This section contains links to all of the course materials for the HIV genotyping exercise.
2. Set the second web browser page by double-clicking on the "HIV protocol" link at http://www.cybertory.org/exercises/HIV_genotyping/protocol.html. It is preferable, however, to provide each student with a paper copy of the protocol prior to performing the exercise to avoid having to change screens to read the instructions in addition to having the web version.
3. Set the third web browser page by double-clicking on the "trace generator" link, it is shown in Figure 2. The URL for the trace generator for the HIV genotyping exercise is at http://www.cybertory.org/exercises under the HIV Genotyping section. It is important that this webpage is used for sequencing the HIV clones as it contains the HIV clones in the pulldown menu and the list of sequencing primers for the HIV genotyping exercise.

The HIV genotyping folder contains an HXB2 genomic reference sequence and a Staden configuration file which is needed to perform the exercise.

1. Go to the first webpage at http://www.cybertory.org/exercises.
2. Click on the "zip archive" link and save the "HIV_genotyping zip archive to your desktop.
3. Unzip the "HIV_genotyping" zip archive and save the HIV_genotyping folder to your desktop.

The instructor should assign each student or pair of students working at a computer an HIV clone from the list of clones in the clone catalog at http://www.cybertory.org/exercises/HIV_genotyping/clone_catalog.html

Each HIV clone has a drug treatment history and associated drug resistance mutations. The clones have been constructed from the complete HXB2 genome (accession #NC_001802) by adding resistance mutations into the HIV protease and RT coding regions based on actual sequence data files from the Stanford HIV database. The treatment history of antiretroviral drugs for each clone are listed in the clone catalog.

Each student or student pair should have a viral "unknown" assigned to them. It is recommended that the instructor walk the students through the sequencing of HIV protease step-by-step using a computer and overhead projection for the first part of the exercise, if possible.

The primers proF and proR sequence the entire HIV protease coding region of 99 amino acids, on both strands, as shown in Figure 1. ProF is the forward sequencing primer and ProR is the reverse sequencing primer. Follow the instructions to sequence HIV protease for the HXB2 reference sequence using the Cybertory^TM trace generator.

1. Go to the third Cybertory web brower, which is the sequence trace generator at http://www.cybertory.org/exercises under the HIV Genotyping section of the webpage.
2. Type in a name in the "User Name" box. For example, use your first or last name or email address (without the @ symbol).
3. From the "Template" pulldown menu, select "HXB2", which is at the bottom of the pulldown list. The list also contains all of the unknowns, which will be sequenced later.
4. The primer sequences are available at the bottom of the trace generator webpage and are summarized in Table 1. Copy and paste the proF sequence "AGCCAACAGCCCCACCAG" into the "Primer Sequence" box of the trace generator.
5. Select the "Primer Strand" pulldown menu to forward, as proF is a forward sequencing primer.
6. Leave all of the other fields at their defaults and double-click on the "Run Sequencing Reaction" button.
7. A window will pop up and ask you to save the file. Select "Save"
8. A second window will pop up. Go to the "file name" box, second to the bottom of the window. Change the name of the file from "HXB2-p1t.scf" to "HXB2-proF.scf".
9. Save the file in the "HIV_genotyping" folder on your desktop.
10. Go back to the trace generator webpage to sequence HXB2 with the reverse sequencing primer proR. This will give you sequence coverage on both strands.
11. Copy and paste the proR sequence "GGGCCATCCATTCCT" into the "Primer Sequence" box of the trace generator.
12. Select the "Primer Strand" pulldown menu to reverse as proR is a reverse sequencing primer.
13. Select the "Annealing Temperature" pulldown menu for this primer to 65 instead of 55 for best results. If 55 degrees is chosen, the sequence will be of poor quality.
14. Leave all of the other fields at their defaults and double-click on the "Run Sequencing Reaction" button.
15. A window will pop up and ask you to save the file. Select "Save"
16. A second window will pop up. Go to the "file name" box, second to the bottom of the window. Change the name of the file from "HXB2-q1t.scf" to "HXB2-proR.scf".
17. Save the file in the "HIV_genotyping" folder on your desktop.
18. Repeat the entire process for your assigned "unknown" for both proF and proR sequencing primers. The unknown is selected in the "Template" pulldown menu instead of HXB2.

We summarize the function and use of each Staden module used in the exercise, including step-by-step instructions on how to use each module.

The trev (trace viewer) application is used for visual inspection of trace files generated by the cybertory trace generator. It is important to examine the quality of the sequence traces before proceeding in order to see that they are of good quality. There should be a single major peak at each position in the trace file.

1. Launch the Trev application by going to the "Start" Menu, select "Programs", "Staden Package" and finally "Trev". This will launch the Trev application.
2. Select the "File" pulldown menu to "Open" to load the trace file from the "HIV_genotyping folder on the desktop. Please note that the "files of type" dialog box at the bottom of the screen must be selected to ".scf" files to view the traces produced by the trace generator. It is set to ".abi" file type by default, and the ".scf" files will not be listed unless the file type is changed.
3. On the top of the sequence traces is a set of arrows and a scroll box that can be used to view the entire trace file. The student is then able to decide if the sequence trace is of sufficient quality; if not they can resequence the template after altering the parameters in the trace generator.

The pregap4 module processes the .scf files created by the trace generator according to a set of parameters which have been selected and defined by a configuration file for the HIV genotyping exercise. Some of the functions of pregap4 are to edit poor quality sequence and vector sequences in the trace file so they will be excluded in the assembly in gap4.

1. Go to the "HIV_genotyping" folder on the desktop and double-click on the configuration file pg4.config. This launches the pregap4 utility with the proper settings for the exercise.
2. Select the "Files to Process" tab at the top of the screen and then select the "Add files" tab. The bottom "Files of type" pulldown menu needs to be .scf instead of .abi.
3. From the HIV-genotyping folder on the desktop, select the four sequence trace files for HIV protease which were sequenced using the trace generator; HXB2-proF, HXB2-proR, clone#-proF and clone#-proR into pregap4.
4. Click on the "Open" button to load the files into pregap4. The files should appear in the main window.
5. Select the "Add files" tab again. The bottom "Files of type" pulldown menu needs to be the last option "any". Select the HXB2.embl reference sequence.
6. Click on the "Open" button to load the file into pregap4.
7. At the top of the pregap4 screen, select the "Configure Modules" tab. Select the "reference traces and sequences" box.
8. To the right of "Reference Trace{+ve strand}" click on the Browse button. Go to the HIV-genotyping folder on the desktop and select "HXB2-proF" as the forward reference trace.
9. To the right of the "Reference Trace{-ve strand}" click on the Browse button. Go to the HIV-genotyping folder on the desktop and select "HXB2-proR" as the reverse reference trace.
10. To the right of "Reference Sequence" click on the Browse button and select "HXB2.embl" as the reference sequence.
11. Process the traces by clicking the "run" button on the lower left hand side of the screen.

This software allows the student to view the sequence trace files in an assembly format. A gap4 database is created after processing the trace files in pregap4 as a ".aux" file.

1. In the "HIV-genotyping" folder on the desktop, locate the "HIV.0.aux file. Double-click on the "HIV.0.aux" file to launch gap4.
2. In the main gap4 window the assembly can be viewed by selecting the "View" pulldown menu, select the "Templates Display" menu option.
3. In the "Show Templates" dialog box select the "all contigs" button and check the "Templates and Readings" boxes. Four sequencing reads (two forward and two reverse) corresponding to the HXB2 control and clone primer sequences are visible in the display window of gap4. The sequence reads should automatically be correctly oriented to the HXB2 reference sequence (HXB2.embl). Orange dots denote differences in sequence between the HXB2 control traces and the clone traces.
4. Double-click on the arrows in the template display to bring up the "Contig Editor" window.
5. In the "Contig Editor" window go to the "Settings" menu and select "trace display" and "autodiff traces".
6. On the left margin of the contig editor window, select the name of the HXB2 reference sequence (HXB2.embl) by double-clicking on it. Right-click to bring up a context menu and select "set as reference sequence." Leave the default options of "first base number" of 1 and "No" for circular sequence and click "ok".
7. An "S" should appear at the far left end of the HXB2 line, denoting HXB2.embl as the reference sequence.
8. To visualize the result of the trace subtraction algorithm, double click on one of the sequence reads in the contig editor (either clone sequence proF or R). Three sets of traces should appear; the HXB2 control trace, the clone trace and a trace subtraction.
9. The trace subtraction will show all of the differences between the clone trace and the HXB2 control trace. These differences correspond to the drug resistance mutations in the clone sequence.
10. To export a list of the mutations as a report, select "Commands" menu and then "Report mutations" from the contig editor’s menu. A plain text version of the mutation report will appear in the gap4 main window. Copy and paste the mutation report from the gap4 main window into a text file.
11. Save the text file as a word document in your desktop folder "HIV_genotyping" as "pro_mutations"

We have written a utility that converts the mutation list from gap4 to a format which can be entered into the Stanford HIV database; it is available under the HIV Genotyping section at http://www.cybertory.org/exercises/.

1. At http://www.cybertory.org/exercises/, scroll down to the HIV Genotyping section and click on the "Reformat the Gap4 mutation report link to bring up the utility that converts your Gap4 mutation list to a list of codons for the Stanford HIV database
2. Copy and paste the mutation report from gap4 into the window of the utility and click on the "Reformat for HIVdb" button. A list of amino acid changes will be produced, which can be copied and pasted into the Stanford HIV database to determine the drug resistance profile of the clone.
3. The amino acid changes are entered into the Stanford HIV database. Go to the sequence analysis section (HIVdb) of the Stanford HIV database: http://hivdb.stanford.edu/
4. Copy and paste the list of amino acid changes into the menu for HIV protease.
5. Click the "analyze" button and the database will generate a mutation report describing sensitivity or resistance to various HIV drugs

The HIV reverse transcriptase (RT) enzyme is larger than HIV protease. RT must be sequenced by three pairs of primers (rt1Fand R, rt2F and R, and rt3F and R) which cover the RT coding region up to codon 333 (Fig. 1). Each primer pair for rt1, 2 and 3 is analyzed separately, using the method described above for sequencing HIV protease. Sequence the clone using the first primer pair, rt1F and R instead of proF and proR, following the instructions with the following exceptions:

All RT primers are sequenced at 55 degrees and follow the naming conventions as described for sequencing HIV protease, for example, HXB2-rt1F, HXB2- rt1R, clone#-rt1F, clone#-rt1R, etc.

Follow steps 1-11 of the pregap4 instructions substituting HXB2-rt1F, HXB2- rt1R, clone#-rt1F, clone#-rt1R for HIV protease. To analyse sequence data from each RT primer pair, create a new gap4 database for each pair.

In the "Configure Modules" section after completing pregap4 setup, select the "Gap4 shotgun assembly" option with the mouse. On the right hand side of the window in the Gap4 database name box type rt1. Select the "create new database" option directly below. Leave the Gap4 database version set to 0. For rt2 and rt3 primer pairs, do a separate pregap4 analysis for each primer pair and name each gap4 database rt2 andrt3, respectively.

Follow the instructions for gap4 analysis. In step 11, for each RT primer pair, save the mutation list as "rt1_mutation" for the rt1F and R gap4 analysis; for rt2F and rt2R, "rt2_mutation" and for rt3F and rt3R, "rt3_mutation". Remember to do a separate pregap4 and gap4 analysis for each primer pair, creating separate gap4 databases rt2 and rt3 for each respective primer pair.

The mutation list from all the primer pairs are copied and pasted together into a single text file to be submitted to the Stanford HIV database to obtain a resistance report.

Follow the instructions for submitting mutations to the Stanford HIV database. Be sure to copy and paste the HIV reverse transcriptase mutations in the HIV RT section of the webpage.

G1. List the experimental parameters that affect the quality of a sequencing reaction.

• What experimental parameters affect the quality of a sequencing reaction?
• Why is it so important to address one variable at a time when you are optimizing a reaction?
• Why is primer design so difficult for HIV sequencing?

G2. Describe the causes of failure of sequencing reactions by relating reaction conditions, primer design and template quality to the trace output from the virtual sequencer.

• What is a hairpin loop and how does it affect a sequencing reaction?
• How is the sequence trace affected if a primer binds to more than one site on a DNA template?
• How is the sequence trace affected if either the primer or the DNA template is not of sufficient purity?
• What is Tm and why do GC base pairs raise the Tm more than AT base pairs in a sequence?
• What would be the most likely reason a sequencing primer would fail in a clinical assay for HIV genotyping?

G3. Describe the primary functions of pregap4 and gap4 in the analysis of sequence data.

• What are the basic processing steps that pregap4 performs on sequence trace files?
• Why is it necessary to run pregap4 before gap4 is run?
• What functions of gap4 are used in this exercise?

G4. Explain the concept of trace subtraction and it's application to HIV genotyping.

• Why do we need the HXB2 reference traces for trace subtraction?
• Why do we need a reference sequence for mutation detection?
• What causes the trace subtraction algorithm to record a peak even when there is no change in nucleotides from the reference trace?

G5. State the difference between mutations that cause drug resistance, those that cause codon changes in the protein sequence and silent mutations which do not cause changes in the protein sequences of HIV protease or RT.

• What is a silent mutation?
• How could a silent mutation or a mutation that caused a codon change negatively affect the HIV genotyping assay?
• Why was it important to use a multiple alignment of HIV sequences when designing the primers for the HIV genotyping assay?

G6. Describe the Stanford HIV drug resistance database.

• What are the two types of ways in which genetic variation occurs in HIV?
• What are the two ways in which you can obtain a resistance report using sequence data?
• There are three major sections to the database, what are they and which one will be most useful for this exercise?