QualTrace DNA sequencing software users manual
The QualTrace™ DNA sequencing software analyzes trace files collected on the ABI 3730 and 3730xl DNA sequencers. The software can detect nine common sequencing sample problems that limit the read length of sequenced DNA. The software generates a text log file reporting the classification of each trace, together with an estimate of the signal to noise ratio at the early, mid, and late trace regions.
The QualTrace software can process either single trace files or complete folders of trace files. If a user specifies an input folder then all AB1 files found within the folder are analyzed. Trace files contained within sub-folders are not analyzed, that is, no file recursion is performed.
The QualTrace log file contains a record of the analysis process in tab delimited format. The log file contains the following fields:
- QT DATE (QualTrace run time)
- FILE (trace file name)
- QT CLASS (QualTrace classification of the trace file)
- QT CODE (QualTrace classification number)
- MACHINE (ABI3730 machine name)
- RUN (ABI run name)
- RUN DATE (ABI run time)
- TRAY WELL (Sample plate well)
- CAPILLARY (ABI3730 capillary number)
- DYE SIGNAL (likelihood of dye peaks or dye blobs)
- EARLY SIGNAL (average signal to noise value from the early region of trace)
- MID SIGNAL (average signal to noise value from the middle region of the trace)
- LATE SIGNAL (average signal to noise value from the late region of trace)
If a QualTrace log file already exists then the new run messages are appended to the existing log file. The default log file name is qt.log, but a user may change this name using the -l option. The -nl option prevents the QualTrace software from writing to the log file. At the end of each analysis run a summary table is written to the log file. detailing the percentage and total counts of traces that fall into each classifications.
A estimate of the likelihood of dye peaks or dye blobs being present in the trace is reported in the DYE SIGNAL field. Values of less than 1 suggest that no dye blob is present, with values between 1 and 5 that a moderate dye blob problem exists, and values above 5 indicating that the trace has very large dye blob present. As a general rule the higher the DYE SIGNAL value the worse the dye blob problem is.
Using the QualTrace DNA sequencing software
To use the windows version of the QualTrace software, open a Windows command shell and enter QualTrace.exe using the desired command line parameters and options. To open a command shell select RUN from the Windows Start Menu and type cmd then press the OK button. An example of what to type in the command shell is shown below:
QualTrace.exe -i C:\inputfolder -l c:\my documents\userlog.log –j 2
QualTrace command line switches and parameters
help -h
Provides the help information for the QualTrace software.
version -V
Provides the version number of the QualTrace software.
input -i [file name | folder name]
Specifies the input file or folder. If a folder is specified then all the ab1 trace files within the folder are processed. Traces contained within sub folders are not processed.
log name -l [log file name]
Specifies the name of the processing status log file This log is always appended to rather than written over.New entries are always appended to the existing log file
The default log file name is qt.log, however, both the log name and path can be user defined.
- UNIX/ Linux: -l /usr/mylogs/userlog.log
- Windows: -l c:\my documents\userlog.log
no log -nl
Disables all writing to the log file
Default is OFF.
multi-processor -j [integer]
Allows the QualTrace software to run multiple sub-processes. This allows multiple traces from an input folder to be process in parallel. This setting is of utility on multi-processor machines or cluster arrays. On single processor machines it is not recommended that more than one sub-process be used.
Default is ONE.
QualTrace analysis codes
Good 0
Indicates that no problems were identified. Alignable Q20+ bases are usually high to very high.
No signal 1
Indicates that the trace contains very weak or no signal (failed reaction or capillary). Many causes including: no template; loss of sample during clean-up; blocked capillary; or sample contamination with salt. Alignable Q20+ bases are usually very low.
Late Signal Start 2
Indicates that the beginning of the trace peaks is delayed. This test checks whether the start of signal (base 0) has begun later than the normal expected time for base 150. This problem is commonly associated with sample contamination with high levels of salt, protein or template DNA. Alignable Q20+ bases are low to moderate.
Spectral calibration 3
Indicates that the trace file contains significant dye crosstalk is present (>15% in at least one channel). Frequent occurrence of this code indicates that a machine spectral calibration may need to be performed. Alignable Q20+ bases are usually moderate to high depending on the severity of the crosstalk.
Early mixed signal 4
Indicates that the trace contains significant mixed signal before base 350. Commonly caused by primer mis-annealing during sequencing or template amplification. Alignable Q20+ bases are moderate.
Late mixed signal 5
Indicates that the trace contains significant mixed signal after base 350. The usual cause is two of more templates present in the reaction. Alignable Q20+ bases are usually low.
Noisy signal 6
Indicates the trace is weak with poorly resolved and/or mixed signal. Many causes including: incorrect template concentration; excessive sequencing chemistry dilution; machine service issues; or sample contamination with salts, proteins or unlabelled DNA. Alignable Q20+ bases are low to moderate.
End weak 7
Indicates low trace signal before base 700 for 36cm arrays or before base 900 for 50cm arrays. Many causes including: incorrect template concentration; excessive sequencing chemistry dilution; or sample contamination with salts, proteins or unlabelled DNA. Alignable Q20+ bases are moderate to high.
Excellent 8
Indicates that the trace is not signal limited. Correlates strongly with maximum trace read-length. Alignable Q20+ bases are usually very high.
Rapid run not supported 9
Rapid run modules traces are currently not support. Support for rapid run module traces is currently under development.
QualTrace errors
The following errors may occur when attempting to process trace files. The various possible causes of these errors and their corresponding code are described together with possible solutions.
No processed data in file 10
This error may occur if the trace file is corrupt. Remove trace from process list.
No base order in file 11
This error may occur if the trace file is corrupt. Remove trace from process list.
Unlicensed machine 20
This error will occur if the machine that produced the trace file has not been licensed for use with QualTrace. Please contact Nucleics to obtain a license for the machine.
Not enough memory 21
This error may occur if too many applications are running. Close nonessential applications and restart QualTrace.
Can't open file 22
This error may occur if the file does not exists or if the file has incorrect access permission settings. Check if file exists and that you read permission to the input folder.
Can't create file 23
This error may occur if you do not have write permission to the selected output folder or if the hard disk is full. If the hard disk is near full please remove excess files and restart QualTrace.
Can't read file 24
This error may occur if an open file was deleted or moved while being used by QualTrace. Do not delete or move trace files after starting QualTrace.
Can't write file 25
This error may occur if you do not have write permission to the selected output folder or if the hard disk is full. Please remove excess files and restart QualTrace and check if you have write permission to the folder selected.
Invalid file 26
This error may occur if the trace file is corrupt. Remove trace from process list.
No raw data in file 29
This error may occur if the trace file is corrupt. Remove trace from process list.
Unsupported machine 32
This error may occur if processing a trace file produced by a sequencing machine type that QualTrace does not support or if the trace file is corrupt. Remove the trace from process list.
Process was aborted 39
This error will occur if a QualTrace sub-process is stopped explicitly. Do not stop the QualTrace sub-processes.
Processing took too long 40
This error occurs if the trace cannot be analyzed within one minute. This error indicates a program malfunction. Please email our .
Program error 42
This error indicates a program malfunction. Please email our
Technical Support
Nucleics provides expert technical support for the QualTrace DNA sequencing software. Please email any questions or comments about Qualtrace to our