Interpreting Chromatograms & Troubleshooting

Rather than re-creating the wealth of information that already exists on the web for the various domestic and international core sequencing sites, we direct you to a couple of excellent, external sites that will provide you with an extensive array of helpful information on cycle sequencing.

Note

We are only directing you to information about interpreting chromatograms and troubleshooting. Any information contained within those pages that relate to specific service provision by that facility, will not necessarily apply at Micromon.

NUCLEICS

This small Australian company has an excellent web site that provides a guide on How to Troubleshoot DNA Sequencing Problems, including a graphical presentation of the most common problems encountered. A free online version of their software QualTrace is available to assist in the identification of some of the common causes of problems.

Nucleics have also produced a DNA Sequencing System for increasing the read length of sequencing traces collected on the Applied Biosystems genetic analysers. The Long Trace software extracts and reprocesses the raw data contained within each ABI trace file using a series of highly sophisticated algorithms developed by Nucleics. This data reprocessing enables the ABI KB base caller to make significantly better base and quality calls on the trace, resulting in up to a 30% increase in the number of QV20 base calls. There is a free online version of this service for users who would like to trial the software. Up to 10 sequences can be processed by each user per day. Trials at Micromon have shown an increase in the QV20+ base call read of our quality control standard, by at least 10%.

UNIVERSITY OF MICHIGAN

This core facility in the USA is also a very useful resource. In particular, the header sections CHROMATOGRAM INTERPRETATION and TROUBLESHOOTING are excellent guides to understanding the features and characteristics of a chromatogram, and suggestions as to why problems and anomalies have arisen.  In addition, there are sections in the FAQ list detailing the Common Reasons for Sequencing Failure and suggestions as to why Sequencing might work but produce Poor Data.

For novice users, there is a comprehensive guide outlining the fundamental theory behind DNA Sequencing and practical advice on how to Prepare Samples to obtain the best possible sequencing result.