The DNA Sequence Research Committee initiated its second study to evaluate some common protocols for sequencing GC-rich templates to answer questions that were raised in its first study (1,2). Specifically, we questioned whether adding DMSO to sequencing reactions consistently improves results, or if altered thermocycling procedures improved sequencing. In addition, we asked study participants to edit the sequences to determine if manual editing consistently improves sequence accuracy. We presented an initial tabulation of results at the ABRF '97 meeting in Baltimore, and the poster information can be found on the World Wide Web through a link on the ABRF WWW Homepage: http://www.abrf.org.
This year's test sample was a plasmid containing a particularly difficult GC-rich insert, provided by Ed Laufer, Olivia Orozco, and Cliff Tabin of the Department of Genetics at Harvard Medical School (3). Participants were asked to sequence the test sample according to the conditions recommended by the manufacturer and also to add 5% DMSO to the sequencing reaction or to alter the thermocycling conditions. Both non-edited and edited sequences for each condition tested were requested by the Committee.
Samples, along with a sample survey, were mailed to 134 laboratories that offer DNA sequencing as a service. Forty-eight facilities returned 246 sequencing chromatograms, and 230 of these could be aligned with the sample's sequence.
Our initial analysis has been based on counting errors over the length range of 0-600, 0-400, and 0-200 bases. Miscalls, insertions, deletions, and undefined bases (N's) were all considered errors. After sorting the data by edited status or reaction conditions, we have generated some preliminary conclusions. Foremost is the effect of editing. As an example, in this year's study 44 sequences were submitted as non-edited, with an average of 20.8 errors over the range of 0 - 400 bases. Of those, 27 submitted edited data with an average of 7.5 errors from 0-400, an improvement from 94.9% accuracy to 98.1%. Manual editing of the data clearly improves the sequence accuracy.
Most of the data returned was obtained using ABI instruments and software and is reflective of that system's strengths and weaknesses. This test sequence included sequence stretches that are extremely difficult for the TaqFS dye-terminator system to read correctly. However, the electropherogram patterns are reproducible and easily learned through experience; and the improvement in sequence accuracy due to editing clearly shows this. This contrasts with our earlier study by the Nucleic Acids Research Committee (4) on automated sequencing using AmpliTaq dye terminators, which showed that editing did not always improve accuracy and, in many instances, decreased it.
Conclusions regarding the effects of altered reaction conditions are preliminary. The accuracy of non-edited data is improved by adding DMSO to sequencing reactions. For the 0-400 base range addition of DMSO (35 sequences) decreased the average number of errors to 12.0, an improvement to 97% accuracy. DMSO also improves accuracy over 0-200 and 0-600 base ranges. However, the effectiveness of altered thermocycling conditions is unclear because different laboratories use different protocols.
The Committee hopes this presentation of preliminary results from this study will help member laboratories assess methods for sequencing GC-rich templates.
References
1. Adams, P.S., Dolejsi, M.K. , Hardin, S., Mische, S., Nanthakamur, B., Riethman, H., Rush, J., and Morrison, P. (1996) DNA Sequencing of a Moderately Difficult Template: Evaluation of the Results from a Thermus thermophilus Unknown Test Sample. BioTechniques 21, 678.
2. Adams, P.S., Dolejsi, M.K., Hardin, S., Mische, S., Nanthakamur, B., Riethman, H., Rush, J. and Morrison, P. DNA Sequencing of a Moderately Difficult Template: Evaluation of the Results from a Thermus thermophilus Unknown Test Sample and General Survey. http://mbcf.dfci.harvard.edu/abrfdnaseq. Last updated, 10/22/96.
3. Laufer, E., Dahn, R., Orozco, O.E., Yeo, C.-Y., Pisenti, J., Henrique, D., Abbott, U.K., Fallon, J.F. and Tabin, C. (1997) Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation. Nature 386, 366-402.
4. Naeve, C.W., Buck, G.A., Niece, R.L., Pon, R.T., Robertson, M. and Smith, A.J. (1995) Accuracy of automated DNA sequencing: a multi-laboratory comparison of sequencing results. BioTechniques 19, 448-453.
Return to the The ABRF Home Page