Sequencher Troubleshooting

This page describes some problems you may encounter when trying to import files with Phred qualities into Sequencher version 4. For general instructions on importing Phred qualities into Sequencher, click here. Sequencher is a leading tool for contig editing and assembly developed by Gene Codes Corporation.

A number of things can go wrong when trying to import qualities from a Phred-generated .fasta or .fasta.qual file. These include:

• Sequencher gives an obscure error message when trying to import
• Sequencher does not show the qualities, or shows qualities only for some of the imported sequences
• Importing does not import all the sequences in the fasta file
• You cannot open the chromatograms after importing
• The qualities shown in Sequencher do not make sense.
• The sequences have wrong file names (like seq.fasta #1) and no qualities

Some of the most common reasons for these problems are:

• Long file names (especially on Windows when processing ABI 3700 data)
• Sequencher tries to open the wrong trace files (Files are not where they should be)
• The sequence in trace files does not match the sequence in the FASTA files
• User preferences for importing are set to

A detailed description of these problems is given below. For the impatient, here are some guidelines to follow to avoid the common problems:

1. Before analyzing files with Phred, make sure all file names are less than 32 characters long.
2. Avoid using "funny" characters (like spaces, \, /, #, ...) in file names. Ideally, use only letters, numbers, and underscores.
3. When running Phred, make sure to create (a) a single FASTA file, (b) a single FASTA.QUAL file, and (c) SCF files.
4. Make sure that all files created by Phred are either all in the same directory, or in directories that follow the structure required by Consed (FASTA and .QUAL files in a directory called edit_dir, and SCF files in a sister directory called chromat_dir).
5. Make sure that the input files for Phred are NOT in the directory above the .FASTA files (and also not in the chromat_dir or the same directory as the Phred-generated SCf files).

Problem 1: Long file names

Sequencher appears to have problems when trying to import sequences and trace files which have long file names.This is a confusing problem, because Sequencher often does not give any error messages - just some things are wrong after importing. These can include:

• No sequences are imported (or only some, but not all sequences are imported)
• All sequences are imported, but the chromatograms are not available
• All sequences are imported, but they do not have correct qualities. Sometimes, just one or two of the imported sequences seem to have qualities, which may or may not make sense, while other sequences do not have qualities at all.

You can easily reproduce (or eliminate) this problem by renaming files before the Phred analysis.In our tests, the same files were imported without problems when the name (with extension) was 31 characters or less, but not if the names were 32 characters or longer. (This problem may be related to the maximum length of file names on the Classic MacOS).

Keep in mind that Windows may not show you the entire file name - the extension may be hidden, depending on the Explorer settings and the extension. To be safe, keep file names at 27 or fewer characters (to allow for the ".scf", ".ab1" etc. extensions) - shorter is safer.

This seems to be a bug in Sequencher, and we have not found a workaround other then renaming the files before analysis with Phred. Please contact GeneCodes with any comments about this.Unfortunately, it also seems to be a common problem, since the default settings on the ABI 3700 tend to produce very long file names.

Problem 2: Where chromatogram files should be (and where not!)

Sequencher tries to automatically find the chromatogram files that belong to a sequence entry in the FASTA (or FASTA.QUAL) file. Apparently, Sequencher always uses the base calls in the chromatogram file, and the quality scores in the FASTA.QUAL file. Since Sequencher does this without any consistency checks, Sequencher may assign quality scores to the wrong bases! This can happen if:

• Sequencher reads the original ABI files instead of Phred-generated SCF files
• The base calls in the SCF files are different from the base calls in the FASTA and FASTA.QUAL files, for example because the SCF files where generated in a different Phred run with different processing parameters
• The FASTA and/or FASTA.QUAL file were edited.

Furthermore, it seems that Sequencher completely ignores the base calling information in the .phd files, which contain the current base calls.

One more problem can that can make it impossible to import Phred qualities: if the directory above the .FASTA and the .QUAL file contain the original ABI files, then Sequencher will (sometimes) give an error message:

File Error! (10000)

Some kind of error occured while attempting to read from or write to
a disk. Therefore your selected command failed.

If you delete, rename, or move the files in the parent directory, the import works fine.

The suggested directory structure and folder contents is as follows (assuming that your input files are in C:\MyProject\)):

Tip: Spaces in file an folder names often can cause problems. If possible, avoid all spaces and other "funny characters" in file and folder names (that also means you should not save files in your "My Documents" folder!).

You will get the directory structures above if you use the "Process: Consed format" or the "AutoPhred and Analysis" options in InterPhace, version 2.2.3 or newer (older InterPhace versions do not create the extra directory ("phred_made") in "AutoPhred", and can the results may not import well into Sequencher; but you can always move things around by hand, if you need to).

Problem 3: The qualities do not make sense

Sometimes, the qualities shown in Sequencher don't make sense. This is typically a result of problems resulting from long filenames or wrong directory structures, as discussed above.

One common mistake is to run Phred to only create FASTA and quality files, but not SCF (chromatogram) files. Sequencher will read the quality file, and then open the original (ABI etc.) trace files. But the base calls in the ABI files and those made by Phred will be different! Sequencher will not complain about this - it will simply assign quality values to the wrong bases without any warning. If you suspect this is the case, you can look at the .phd files, using a text editor like WordPad. A few lines down in the files, you will see the base calls made by Phred, and the associated qualities and positions in the trace. Typically, the first few bases are already different from those called by the ABI software and shown in Phred.

Note that problem is not limited to ABI, it can happen with other input files, too. You can always use InterPhace or TraceViewer to check if a trace file has Phred qualities by opening the file in TraceViewer or InterPhace: Phred-generated SCF files will show quality bars at the top, ABI files will not.

If you see this problem, simply re-process the files with Phred, making sure to create the correct directories, and then import the newly create files.

Problem 4: Wrong file names and no qualities

Some users have experience the following problem: after importing sequences from Phred-generated FASTA files, the file names of all sequences are wrong, and the sequences do have qualities (no colored backgrounds). The names of the imported sequences are often like "seq.fasta #1", "seq.fasta #2), and so on.

A related problem is that Sequencher does not import anything from Phred-generated quality (.qual) files.

To fix this problem, do the following:

1. Open Sequencher.
2. Select "User Preferences..." in the "Windows" menu.
3. If necessary, click on the triangle before "Input/Output" on the left side so that you can see the different input/output preference options.
4. Click on "File Import" on the left side.
5. Make sure that the checkbox before "Prefer File Names to Data Names" in the "Import Options" section is not checked.
6. Close the User Preferences window.
7. Try importing the same file again. Importing should work fine now.
8. If it you still have the same problem: please check the other problem descriptions above, especially the "long file names" problem. Also, double-check your import preferences as described above.