1. Create a new folder on the desktop called “Trace” (right click on desktop, > new> folder, and then right click on the folder, > rename > type in Trace) and copy sequence files ending with .phd.1 and .ab1 from the Trace Package that LIMS sent you into the new folder. Do not copy the blanks.
  2. Go to the start menu (bottom left corner of the desktop) and select Run… and in the box “Open:” type “cmd” and click ok.
  3. A window will open with a black background, this is the command prompt window. Type “cd desktop” (without quotation marks and note the space between cd and desktop)
  4. Press Enter.
  5. Type “cd Trace” (without quotation marks and note the space between cd and Trace).
  6. Press Enter.
  7. It should now say something like C:\Documents and Settings\Hanner-lab\Desktop\Trace> this represents that you are in the trace file.
  8. Type “dir > list.txt” (without the quotation marks and note the space before and after the > sign)
  9. Press Enter.
  10. It should look something like this if all worked:
C: Documents and Settings\Hanner Lab>cd trace
C: Documents and Settings\Hanner Lab\Desktop>cd desktop
C: Documents and Settings\Hanner Lab\Desktop\Trace>dir > list.txt
C: Documents and Settings\Hanner Lab\Desktop\Trace
  1. If it worked, there will not be an error message. This has just created a new file in Trace called list.txt.
  2. Close the command prompt window.
  3. Open Excel, and go to File > open - change Files of Type to All files (*.*) > Desktop > Trace > list.txt > open.
  4. A window will come up called Text Import Window. Click next. Scroll down until you can see your first file named .phd.1 or .ab1 and move the arrowed line beside it over to the right a little so that it touches the file name.
  5. Click next. Click Finish.
  6. You should now have an excel document with a lot of information, and one column listing your files that are alternating phd.1 and .ab1 . These file types have to be sorted. In the cell right to the first file name, type in the formula =right(F8, 3) and press Enter. This formula might have E8 instead of F8 depending, but you want to chose the letter for the column that has your files listed in it. Drag the formula down so that there is a list of ab1 and d.1 in the column beside your file names.
  7. Press Control A to highlight the page, then go to Data > Sort and then chose to sort it by the column with d.1 and ab1 in it. Click ok. Your file names should now be sorted by their endings.
  8. Open a new Excel file and set it up with the following headings:
Trace File Score File Fwd Rev Seq Primer Read Direction Process ID

  1. Go back to the list you just sorted in the excel sheet list.txt and copy the entire list into the first column called |Trace File| (from column F to column A of the new file).
  2. Select the .phd.1 files and cut them (control X) and past them into the column called |Score File|
  3. In the |Fwd| column enter the registered name of the forward primer used during the PCR reaction (for fish this is C_FishF1t1). Copy it down through the entire column to the end of your file lists.
  4. In the |Rev| column enter the registered name of the reverse primer used during the PCR reaction (for fish this is C_FishR1t1). Copy it down through the entire column to the end of your file lists.
  5. In the column |Seq Primer| enter the registered name of your sequencing primer. Copy it down through the entire column to the end of your file lists. It will alternate between forward and reverse. For example, M13F should line up with the read direction Forward. M13F is the sequencing primer used for tailed reactions, like for mammals and fish.
  6. In the |Read direction| enter the names Forward or Reverse depending on the direction of the sequence files it refers to.
  7. In the |Process ID| you need to type in the formula =left(A2, 11) where A2 is the column with your first .ab1 file and 11 characters is for the process ID. For example CWSHK143-08 has 11 characters.
  8. Press Control A to select the entire page. Press Control C to copy the page, and then go Edit > Paste Special and chose values and press OK. This removes the formulas from your page.
  9. Save this file excel spread sheet under the name data.xls and save it in your folder Trace.
  10. Close Excel but do NOT save changes made to list.txt.
  11. Delete list.txt from your Trace folder.
  12. Right click on the Trace folder Send to > Compressed (zipped) folder
  13. This will create a folder with a zipper on the front.
  14. Go to BOLD, log in, and find the project that you want to upload trace files to.
  15. Under uploads, find Trace Files. Browse until you find your zipped trace file. Press submit.
  16. Delete the Trace folders but save the Trace Files somewhere on the computer.

Return to Barcoding in the Hanner Lab Wiki.

Updated May 2 2009