Common Problems

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Phred "Unknown Chemistry"

Phred on Windows

Phred Parameter File Problems

This page describes a common "unknown chemistry" problem when running Phred, which leads to a error message like this:

  /Documents/test_sample_001.ab1: unable to match primer ID string: skipping chromatogram
    unknown chemistry (DT3700POP5{BDv3}.mob) in chromat /Documents/test_sample_001.ab1
    add a line of the form
    "DT3700POP5{BDv3}.mob"    <chemistry>    <dye type>   <machine type>
    to the file /usr/local/genome/lib/phredpar.dat
    type `phred -doc' for more information

Many users encounter this problem; fortunately, it is very easy to fix. You may encounter this problem again in the future, when ABI releases a new chemistry, so will give you a little bit of background information first.

Please note that Phred will also issue error messages everytime Phred tries to read a file that is not a chromatogram file. For example, if your ABI input folder contains text (.seq) files, you should get an error message for each of these. Look closely at the error message - if it is a file (or folder) that is not a chromatogram file, you can ignore it.

What's going on?

When Phred starts processing a file, Phred first tries to determine some things it needs to know about the chromatogram:

• What chemistry (dye terminator or dye primer) was used?
• What dye type was used? (for example rhodamine or energy-transfer)
• What kind of DNA sequencer does this sequence come from? (for example ABI377, ABI3700, or MegaBACE)

Phred gets this information by first reading a short string in the input file, the "primer ID string", and then matching this primer ID string with entries in the "Phred parameter" file. The Phred parameter file has lines like this:

"DP6%25Ac{-21M13}"   primer      rhodamine         ABI_373_377
"ET Terminators"     terminator  energy-transfer   MolDyn_MegaBACE

So, if Phred finds a primer ID string "DP6%25Ac{-21M13}", Phred now knoes that this was dye primer chemistry with rhodamine dyes, run on an ABI 373 or 377 sequencer.

Phred then adjust the base calling for the given machine, and uses a machine-and chemistry-specific lookup table for quality values. This allows Phred to come up with high accuracy for base calls and quality scores.

If Phred cannot find an entry for a given primer ID string in the Phred parameter file, Phred does not know what to do with a sequence, so it gives the error message shown above, and ignores the file. Typically, all your files will be done with the same chemistry, so you will get an error message for every file! But you can help Phred and fix this problem in less than five minutes, at described in the next section.

How to fix it

All you need to do is add a line for the "primer ID string" to your Phred parameter file with any text editor. The Phred error mesage tells you what the line should look like, and where the Phred parameter file is. If you need more help, read on. We will tell you where the Phred parameter file is, which program to use to edit the file, and what potential problems you may encounter.

Where is the "Phred parameter file"?

That depends on your operating system, and where you installed the software. The error message tells you the name and location of the file:

    to the file /usr/local/genome/lib/phredpar.dat

Here, the file is called "phredpar.dat", and is in /usr/local/genome/lib (the default location for OS X, UNIX, and Linux). The following table shows the default names and locations for different operating systems:

If you installed the software at a different location, your file may be in a different folder; again, check the error message!

Please not that you may not see the ".dat" extension on Windows and OS X (depending on your system settings - if you have "Hide extensions of know file type" selected).

Which program should I use to edit the file?

You can use any text editor like NotePad on Windows or emacs on UNIX. Some programs may be problematic because they prefer to save files in binary format or "Rich Text Format". Some suggested programs are:

Open the program you want to use, and then use "Open..." in the "File" menu to open the Phred parameter file. Find a line that is close to what you want; copy and paste it; and then edit the line so that it exactly shows the "primer ID string" in the Phred error message. Then save the file (always save as "Plain text" if your editor gives you several options).

Here's an example, based on the error message above. Phred says to add a line:

    "DT3700POP5{BDv3}.mob"    <chemistry>    <dye type>   <machine type>

You should know the chemistry, dye type, and machine; if in doubt, as the people who did the sequencing. The name here indicates terminator ("DT"), big dye (BD) chemistry from ABI3700. In the Phred parameter file, we find a very similar line:

"DT3700POP5{BDv3}v1.mob"	terminator	big-dye	                ABI_3700

After copy, paste, and editing, we now have two lines like this:

"DT3700POP5{BDv3}v1.mob"	terminator	big-dye	                ABI_3700
"DT3700POP5{BDv3}.mob"	terminator	big-dye	                ABI_3700

If Phred does not know about your sequencing machine type, use the one that is most similar. For example:

• for ABI 3730 or 3730XL data, use ABI_3700 as the machine type
• for ABI 310 data, use ABI_3100 as the machine type

Save the file after making your changes, and test it by running Phred again- the Phred error message should now be gone. If you still have problems, read on to the following section.

I'm still having problems!

On Linux, UNIX, and OS X, you may have permission problems when trying to edit or save the file. To get around this, change the permissions of the file so that everyone can write to it. You may need to ask your system administrator to do this for you, and you may also want to change permissions back after editing the file if you do not want anyone else to change it.

If you encounter other problems, please do the following:

1. Check the spelling - did you enter the primer ID string exactly as in the Phred error message?
2. Did you enclose the primer ID string in quotes, and on a separate line?
3. Did you enter all necessary primer ID strings (you may have to add more than one line)?
4. Did you save the file as plain text? (Word prefers to save in Word (.doc) format, TextEdit in "Rich Text Format", both of which cannot be read by Phred).
5. Did you edit the correct file? (You may have several copies of the Phred parameter file around)
6. Does the file have the correct line endings? (UNIX-style on OS X; should not be a problem on other systems, unless you copied a file from a completely different operating system)

If you did all these checks, and still encounter problems, please contact us. Please include a copy of your Phred parameter file with your email, as well as an exact copy of the Phred error message you get (save it to a file and attach the file, or send a screen shot).

For the curious

If you want to read more about the Phred parameter file, please check the original documentation.

If you encounter problems with Phred being unable to find Phred parameter file on Windows, solutions are described on a separate page.