Improving the Analysis of DNA Sanger Sequencing Results: SeqBase Computer Program

Background. The software provided by the manufacturers of automatic genetic analyzers, in most cases, allows an adequate analysis of the results of Sanger DNA sequencing for templates with a nucleotide composition close to the equivalent. However, to consider the results of sequencing of templates with non-equivalent nucleotide composition, it is necessary to analyze electrophoregrams with preservation of primary information on the intensity of fluorescence signals. This is especially important for the sequencing of DNA modified with sodium bisulfite. Aim: to develop and validate in the practice of scientific research a computer program that ensures adequate analysis of electrophoregrams of Sanger DNA sequencing based on preservation of the primary data and on accurate determination of baselines in the spectral channels of individual nucleotides. Methods. The SeqBase program is written in C#, the programming platform .NET Framework 4.0, and runs in the CLR (Common Language Runtime) for Windows operating systems. SeqBase installation package address is http://www.epigenetic.ru/projects/seqbase. Results. A computer program has been developed designed to analyze the primary results of Sanger sequencing (chromatograms of capillary electrophoresis) obtained from automatic genetic analyzers and presented in files of the ABIF (*.ab1) format, which provides the following functions: 1) viewing the original electrophoregrams both in general form and separately by spectral channels; 2) cropping the area of analysis; 3) signal smoothing; 4) manual setting of the baseline for each of the spectral channels; 5) convergence of baselines on all channels; 6) manual correction of the mobility of DNA fragments depending on the type of fluorescent label of the terminating nucleotide. The program has been successfully tested in a number of studies, the results of which have been published in peer-reviewed scientific journals. Conclusion. The use of the SeqBase program is advisable for the analysis of the results of Sanger sequencing of DNA templates with non-equivalent nucleotide composition, especially those modified with sodium bisulfite, to avoid false results and to clarify quantitative estimates.

DNA Sanger sequencing, bisulfite sequencing, sequenogram analysis, SeqBase computer program

1. Hayatsu H. Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis - a personal account. Proceedings of the Japan Academy. Series B 2008; 84(8): 321-330. doi: 10.2183/pjab.84.321.

2. Frommer M., McDonald L.E., Millar D.S., Collis C.M., Watt F., Grigg G.W. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proceedings of the National Academy of Sciences 1992, 89(5): 1827-1831. doi: 10.1073/pnas.89.5.1827.

3. Abramycheva N.Y., Fedotova E.Y., Nuzhnyi E.P., Nikolaeva N.S., Klyushnikov S.A., Ershova M.V. et al. Epigenetics of Friedreich’s Disease: Methylation of the (GAA) n-Repeats Region in FXN Gene. Annals of the Russian academy of medical sciences 2019; 74(2): 80-87.doi: 10.1038/s10038-019-0696-z.

4. Baldin A.V., Grishina A.N., Korolev D.O., Kuznetsova E.B., Golovastova M.O., Kalpinskiy A.S. et al. Autoantibody against arrestin-1 as a potential biomarker of renal cell carcinoma. Biochimie 2019; 157: 26-37. doi: 10.1016/j.biochi.2018.10.019.

5. Golovastova M.O., Tsoy L.V., Bocharnikova A.V., Korolev D.O., Gancharova O.S., Alekseeva E.A. et al. The cancer-retina antigen recoverin as a potential biomarker for renal tumors. Tumor Biolog. 2016; 37(7): 9899-9907. doi: 10.1007/s13277-016-4885-5.

6. Karandasheva K., Pashchenko M., Tanas A. S., Strelnikov V. V., Kuznetsova E. Improving detection level of somatic mosaicism in neurofibromatosis type 1. Annals of Oncology 2019; 30: v23-v24. doi: 10.1093/annonc/mdz238.083.

7. Карандашева К. О., Пащенко М. С., Дёмина Н. А., Акимова И. А., Макиенко О. Н., Петухов, М. С., с соавт. Соматический мозаицизм при нейрофиброматозе первого типа. Медицинская генетика 2019; 18(5):28-36. doi: 10.25557/2073-7998.2019.05.28-36.