The introduction of fluorescence-based DNA sequencing systems has significantly improved the accuracy and efficiency of DNA sequencing. The use of fluorescence-based systems has led to the development of software to perform gel analysis and assign bases eliminating the time consuming radioactive-based techniques. Although automation has simplified DNA sequencing allowing large amounts of data to be quickly generated and analysis performed, errors still occur in the sequencing process. The researcher should not trust entirely the associated text file. Many issues with sequencing results are not recognized by viewing the text file alone. Sequence data should always be evaluated by revewing the accompanying chromatogram to identify problem data and check base calls made by the analysis software.
This guide highlights common issues associated with sequencing results. A discussion of optimal conditions and the affect of contaminants on sequencing quality is also presented. Using the menubar on the right hand side, navigate to to find information related to specific issues. The DNACF staff are always willing to discuss results and offer suggestions.
Of course, these pages are not a comprehensive review of all sequencing and editing issues that a researcher may encounter but a few of the more common problems that will arise when sequencing. Other resources are available on the web that discuss optimizing your sequence reaction. A good source to begin with is the QIAGEN Guide to Template Purification and DNA Sequencing.