Importing Phred results into Sequencher

This page describes how to import Phred results into Sequencher. Sequencher is a leading tool for contig editing and assembly developed by Gene Codes Corporation. If you encounter problems importing Phred results into Sequencher, please visit the Sequencher troubleshooting page.

As of February 2001, newest Macintosh version of Sequencher (4.1.2) provides functions to import PHRAP-generated assemblies and PHRED quality scores. An example of how macPHRED-MacPHRAP and Sequencher together can give much faster and better assemblies is shown at www.codoncode.com/support/contig_editors.htm#Sequencher.

The Windows version of Sequencher (4.05), however, only offered support for Phred quality scores, but no import functions for PHRAP-generated assemblies; please contact Gene Codes Corporation with questions about release plans.

However, using PHRED quality scores in Sequencher can substantially reduce the efforts in sequencing projects. Quality scores can be used for "end clipping", and typically give better results than older methods based on ambiguities.

Here, we show how to import, view, and use PHRED quality scores in Sequencher.
A note of caution: please always make sure that you import PHRED-generated SCF files when importing into Sequencher! If you do not generate SCF files, or place the FASTA files in the same directory as the original ABI traces, Sequencher will show PHRED quality scores with ABI base calls - which does not make any sense, since ABI and PHRED basecalls almost always differ!

Sequencher for Windows

Sequencher 4.05 for Windows can import Phred quality scores from Phred-generated quality files (".QUAL" files), and associate the quality scores with chromatograms (SCF files) as long as the SCF files are in the same directory as the .QUAL files. The .QUAL files must contain header lines that point to the chromatogram file, like this:

>A060.s CHROMAT_FILE: A060.s
6 6 6 8 9 8 8 8 8 10 13 13 12 9 9 9 18 16 15 13 13 
9 16 20 28 34 29 29 34 36 36 38 38 29 33 25 34 23 
25 17 15 20 20 24 21 21 28 35 20 36 41 35 32 35 35 
51 51 51 51 59 59 59 41 41 25 25 20 20 22 29 28 51 

If you use the programs Phd2fasta or phredPhrap to generate the quality files, the format should be ok. If you are using CodonCode's MacPhred, you may need to update to a new version that generates Sequencher-compatible output files.

Using Phred results in Sequencher for Windows - Step by step

Before starting, gather the chromatogram files you want to analyze together in one folder. We'll assume this folder is named C:\Abi on Windows. Of course, you also need a copy of Phred.

Step 1: Creating input files for Sequencher
Option 1a: From the MS-DOS command line (the hard way) Option 1b: Using InterPhace (the much easier way) Create a new folder for the result files (we'll call it C:\Abi\phredmade) Open an MS-DOS (command line) window. Run phred and create PHD files and SCF files in the new directory: phred -id C:\Abi -cd C:\Abi\phredmade -pd C:\Abi\phredmade Run phd2fasta to create a FASTA file and a quality file from the PHD files you just created: phd2fasta -id C:\Abi\phredmade -os C:\Abi\phredmade\sequence.txt -oq C:\Abi\phredmade\sequence.txt.qual InterPhace is a Java program that provides a graphical "front end" for running Phred on Windows (or Unix). The easiest way to generate Sequencher-compatible quality files is to use the "Process" menu. This will run Phred so that Phred creates .phd (ina sub-directory called "phd_dir) and SCF files (in a subdirectory called "chromat_dir"), and then run phd2fasta, which creates the Sequencher-compatible quality file (in a sub-directory called "edit_dir"). Process will also create a Phrap assembly, which you can ignore (at least until an updated version for Windows with ACE file import functions comes out). For more information about InterPhace, please visit www.codoncode.com/interphace/. You are now ready to import the results into Sequencher.
Step 2: Reading Phred result files into Sequencher
Open Sequencher. Create or open a project, if needed. In the "File" menu, select "Import", then "Import Seqences.." In the "Open" dialog box, move to the folder C:\Abi\phredmade Select "All files (*.*)" in the file type pulldown menu Now select the file "sequence.txt", and click ok. Sequencher should read the file, and show several new icon (one for each sequence) in the project Window. Double-click on one of the icons to open the sequence window. A button at the top right should say "Show Chromatogram". If not, something went wrong.
Step 3: Viewing Phred qualities
Sequencher can display sequence qualities by changing the background behind the bases in text windows. To see this, you may have to change several settings: In the "Windows" menu, select "User Preferences". Select "Confidence" in the "General" section. Enter the number 20 in the left box, and 30 in the right box (you can change these numbers later). It should look like this: Close the "User Preferences" window. Select the "View" menu, and make sure the "Display base confidences" item is checked (select it to add or remove the check mark). Now, open a sequence by double-clicking on an icon in the project window. If everything went well, the sequence should be displayed on a colored background, like this: Darker background colors correspond to lower qualities. You will notice a few low quality (dark) bases at the beginning, and a more lower quality bases towards the end of sequences, where the gel resolution deteriorates. Smaller darker spots in the middle of a sequence can be due to compressions, polymerase stops, or secondary bands. If you have followed the steps above and still have problems importing Phred results into Sequencher, please visit the Sequencher troubleshooting page.
Step 4: Using quality scores for end trimming
Sequencher can use quality scores for end trimming - the removal of low quality bases at the beginning and end of sequences. To try this, select one or more sequence in the project window, and then select "Trim Ends.." in the "Sequence" menu. A window like this will pop up: Click on the "Change Trim Criteria" button, which will pop up a window like this: For starters, make sure that your settings are exactly like shown above. You can play around with different settings later. Click "Ok" in the trim parameter window, and then on the button labeled "Trim Checked Items" in the trim window. A warning dialog will appear, asking you if you really want to apply the clips. After applying the clips, the remaining sequences will be shorter, but only low quality sequence at the ends has been removed. We found that quality-based trimming can give better assemblies - fewer contigs with fewer discrepancies.

Try it!

If you have Sequencher for Windows, and you'd like to try how it works with quality scores, here are some suggestions:

1. Download an example ZIP file (phredmade.zip, 293K) - a collection of Phred-generated sequences for import into Sequencher.
2. Open Sequencher, and import the file named "sequence.qual" (see Step 2 above).
3. Select all sequences, and press "Assemble automatically". This will show you how the assembly works without end trimming (not so well - only two matches are assembled with default assembly parameters).
4. Select all contigs, and then "Dissolve contigs" in the "Contig" menu. You'll get two warning dialog boxes, click "Ok" in the first and then "Dissolve contigs" in the second.
5. Try viewing Phred qualities as described in Step 3 above.
6. Perform a quality-based end trimming as described in Step 4 above.
7. Now click "Assemble automatically" again. This time, Sequencher should assemble everything into one contig.

If you want to try this with your own data, and don't have Phred yet, you can download trial versions from our web site; please visit www.phrap.com/download.htm.