Difference between revisions of "APe Sequence Analysis"
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* Under the “Tools” menu, choose “Align Sequences…”. Choose your receiving plasmid.seq as the reference, using the drop down menu. In the “Align to Windows” field, select any comparison .seq files by clicking on the file names. Click on the button “Show Alignment Parameters and set them as shown. This will cause your sequence to not be split into small fragments. Click OK.<br> | * Under the “Tools” menu, choose “Align Sequences…”. Choose your receiving plasmid.seq as the reference, using the drop down menu. In the “Align to Windows” field, select any comparison .seq files by clicking on the file names. Click on the button “Show Alignment Parameters and set them as shown. This will cause your sequence to not be split into small fragments. Click OK.<br> | ||
<center> | <center> | ||
| − | [[File: | + | [[File:align_options.png]] |
</center> | </center> | ||
* In the resulting window, you should be able to find the newly cloned DNA if GGA was successful. | * In the resulting window, you should be able to find the newly cloned DNA if GGA was successful. | ||
| − | * Now you want to open the matching chromat file .ab1. Adjust the X and Y sliders so that the peak heights nearly reach the letters and the spacing between peaks is big enough to clearly see each peek (see below). You can use this to confirm or clarify any bases that do not match your expectations. | + | * Now you want to open the matching chromat file .ab1. Adjust the X and Y sliders so that the peak heights nearly reach the letters and the spacing between peaks is big enough to clearly see each peek (see below). You can use this to confirm or clarify any bases that do not match your expectations. You can see the Phred scores for each base in the screen shot below. |
<center> | <center> | ||
| − | [[File: | + | [[File:2chromats.png|800px]] |
</center> | </center> | ||
| + | * You can use the "Find" function to locate the bases that flank the questionable nucleotides. Look at the chromatogram and determine what the base should be. | ||
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Latest revision as of 13:12, 28 September 2022
ApE Software for DNA Sequence Analysis
- Download the free ApE software if you want it on your own computer. All college-owned computers already have ApE installed so you can use those computers if you prefer. This tool is used by researchers all over the world.
- Access your sequence files. Click once on a file with the .seq suffix. For Macintosh computers, hold down the command button and type i. A window will open. About halfway down the window under the heading "Open With", choose ApE as the application. Then click on the "Change All" button so that all .seq files will automatically open with ApE.
- Repeat the previous directions but use one of your .ab1 file instead of .seq file.
- Download the 4 receiving plasmids and identify which plasmid you used. Open the correct .seq that was your receiving plasmid.
- Open the appropriate .seq file for your eXperimental samples.
- When open your .seq files, you will see some N bases. The software inserts an N whenever it cannot tell for sure which base should go there. The software that “calls” each base uses three criteria: phred score of quality; width of the peak; and spacing between peaks. Phred scores can be seen as the light bar graphs behind the colored lines. Good scores result in a bar graph about half way up the sequence box. Each base is associated with a different color line.
- Under the “Tools” menu, choose “Align Sequences…”. Choose your receiving plasmid.seq as the reference, using the drop down menu. In the “Align to Windows” field, select any comparison .seq files by clicking on the file names. Click on the button “Show Alignment Parameters and set them as shown. This will cause your sequence to not be split into small fragments. Click OK.
- In the resulting window, you should be able to find the newly cloned DNA if GGA was successful.
- Now you want to open the matching chromat file .ab1. Adjust the X and Y sliders so that the peak heights nearly reach the letters and the spacing between peaks is big enough to clearly see each peek (see below). You can use this to confirm or clarify any bases that do not match your expectations. You can see the Phred scores for each base in the screen shot below.
- You can use the "Find" function to locate the bases that flank the questionable nucleotides. Look at the chromatogram and determine what the base should be.
