iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC147367
Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome - PMC Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Feb 15;26(4):967–973. doi: 10.1093/nar/26.4.967

Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome.

M J Rieder 1, S L Taylor 1, V O Tobe 1, D A Nickerson 1
PMCID: PMC147367  PMID: 9461455

Abstract

Diagnostic re-sequencing plays a central role in medical and evolutionary genetics. In this report we describe a process that applies fluorescence-based re-sequencing and an integrated set of analysis tools to automate and simplify the identification of DNA variations using the human mitochondrial genome as a model system. Two programs used in genome sequence analysis (Phred, a base-caller, and Phrap, a sequence assembler) are applied to assess the quality of each base call across the sequence. Potential DNA variants are automatically identified and 'tagged' by comparing the assembled sequence with a reference sequence. We also show that employing the Consed program to display a set of highly annotated reference sequences greatly simplifies data analysis by providing a visual database containing information on the location of the PCR primers, coding and regulatory sequences and previously known DNA variants. Among the 12 genomes sequenced 378 variants including 29 new variants were identified along with two heteroplasmic sites, automatically detected by the PolyPhred program. Overall we document the ease and speed of performing high quality and accurate fluorescence-based re-sequencing on long tracts of DNA as well as the application of new approaches to automatically find and view DNA variants among these sequences.

Full Text

The Full Text of this article is available as a PDF (322.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson S., Bankier A. T., Barrell B. G., de Bruijn M. H., Coulson A. R., Drouin J., Eperon I. C., Nierlich D. P., Roe B. A., Sanger F. Sequence and organization of the human mitochondrial genome. Nature. 1981 Apr 9;290(5806):457–465. doi: 10.1038/290457a0. [DOI] [PubMed] [Google Scholar]
  2. Bidooki S. K., Johnson M. A., Chrzanowska-Lightowlers Z., Bindoff L. A., Lightowlers R. N. Intracellular mitochondrial triplasmy in a patient with two heteroplasmic base changes. Am J Hum Genet. 1997 Jun;60(6):1430–1438. doi: 10.1086/515460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cann R. L., Stoneking M., Wilson A. C. Mitochondrial DNA and human evolution. Nature. 1987 Jan 1;325(6099):31–36. doi: 10.1038/325031a0. [DOI] [PubMed] [Google Scholar]
  4. Chee M., Yang R., Hubbell E., Berno A., Huang X. C., Stern D., Winkler J., Lockhart D. J., Morris M. S., Fodor S. P. Accessing genetic information with high-density DNA arrays. Science. 1996 Oct 25;274(5287):610–614. doi: 10.1126/science.274.5287.610. [DOI] [PubMed] [Google Scholar]
  5. Cheng S., Higuchi R., Stoneking M. Complete mitochondrial genome amplification. Nat Genet. 1994 Jul;7(3):350–351. doi: 10.1038/ng0794-350. [DOI] [PubMed] [Google Scholar]
  6. Comas D., Päbo S., Bertranpetit J. Heteroplasmy in the control region of human mitochondrial DNA. Genome Res. 1995 Aug;5(1):89–90. doi: 10.1101/gr.5.1.89. [DOI] [PubMed] [Google Scholar]
  7. Eng C., Vijg J. Genetic testing: the problems and the promise. Nat Biotechnol. 1997 May;15(5):422–426. doi: 10.1038/nbt0597-422. [DOI] [PubMed] [Google Scholar]
  8. Gill P., Ivanov P. L., Kimpton C., Piercy R., Benson N., Tully G., Evett I., Hagelberg E., Sullivan K. Identification of the remains of the Romanov family by DNA analysis. Nat Genet. 1994 Feb;6(2):130–135. doi: 10.1038/ng0294-130. [DOI] [PubMed] [Google Scholar]
  9. Hacia J. G., Brody L. C., Chee M. S., Fodor S. P., Collins F. S. Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis. Nat Genet. 1996 Dec;14(4):441–447. doi: 10.1038/ng1296-441. [DOI] [PubMed] [Google Scholar]
  10. Horai S., Hayasaka K., Kondo R., Tsugane K., Takahata N. Recent African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):532–536. doi: 10.1073/pnas.92.2.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Horai S., Murayama K., Hayasaka K., Matsubayashi S., Hattori Y., Fucharoen G., Harihara S., Park K. S., Omoto K., Pan I. H. mtDNA polymorphism in East Asian Populations, with special reference to the peopling of Japan. Am J Hum Genet. 1996 Sep;59(3):579–590. [PMC free article] [PubMed] [Google Scholar]
  12. Ivanov P. L., Wadhams M. J., Roby R. K., Holland M. M., Weedn V. W., Parsons T. J. Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nat Genet. 1996 Apr;12(4):417–420. doi: 10.1038/ng0496-417. [DOI] [PubMed] [Google Scholar]
  13. Jaksch M., Gerbitz K. D., Kilger C. Screening for mitochondrial DNA (mtDNA) point mutations using nonradioactive single strand conformation polymorphism (SSCP) analysis. Clin Biochem. 1995 Oct;28(5):503–509. doi: 10.1016/0009-9120(95)00035-8. [DOI] [PubMed] [Google Scholar]
  14. Ju J., Glazer A. N., Mathies R. A. Energy transfer primers: a new fluorescence labeling paradigm for DNA sequencing and analysis. Nat Med. 1996 Feb;2(2):246–249. doi: 10.1038/nm0296-246. [DOI] [PubMed] [Google Scholar]
  15. Keightley J. A., Hoffbuhr K. C., Burton M. D., Salas V. M., Johnston W. S., Penn A. M., Buist N. R., Kennaway N. G. A microdeletion in cytochrome c oxidase (COX) subunit III associated with COX deficiency and recurrent myoglobinuria. Nat Genet. 1996 Apr;12(4):410–416. doi: 10.1038/ng0496-410. [DOI] [PubMed] [Google Scholar]
  16. Kogelnik A. M., Lott M. T., Brown M. D., Navathe S. B., Wallace D. C. MITOMAP: a human mitochondrial genome database. Nucleic Acids Res. 1996 Jan 1;24(1):177–179. doi: 10.1093/nar/24.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kwok P. Y., Carlson C., Yager T. D., Ankener W., Nickerson D. A. Comparative analysis of human DNA variations by fluorescence-based sequencing of PCR products. Genomics. 1994 Sep 1;23(1):138–144. doi: 10.1006/geno.1994.1469. [DOI] [PubMed] [Google Scholar]
  18. Larsson N. G., Clayton D. A. Molecular genetic aspects of human mitochondrial disorders. Annu Rev Genet. 1995;29:151–178. doi: 10.1146/annurev.ge.29.120195.001055. [DOI] [PubMed] [Google Scholar]
  19. Lee L. G., Spurgeon S. L., Heiner C. R., Benson S. C., Rosenblum B. B., Menchen S. M., Graham R. J., Constantinescu A., Upadhya K. G., Cassel J. M. New energy transfer dyes for DNA sequencing. Nucleic Acids Res. 1997 Jul 15;25(14):2816–2822. doi: 10.1093/nar/25.14.2816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marzuki S., Noer A. S., Lertrit P., Thyagarajan D., Kapsa R., Utthanaphol P., Byrne E. Normal variants of human mitochondrial DNA and translation products: the building of a reference data base. Hum Genet. 1991 Dec;88(2):139–145. doi: 10.1007/BF00206061. [DOI] [PubMed] [Google Scholar]
  21. Metzker M. L., Lu J., Gibbs R. A. Electrophoretically uniform fluorescent dyes for automated DNA sequencing. Science. 1996 Mar 8;271(5254):1420–1422. doi: 10.1126/science.271.5254.1420. [DOI] [PubMed] [Google Scholar]
  22. Nickerson D. A., Tobe V. O., Taylor S. L. PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res. 1997 Jul 15;25(14):2745–2751. doi: 10.1093/nar/25.14.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Parsons T. J., Muniec D. S., Sullivan K., Woodyatt N., Alliston-Greiner R., Wilson M. R., Berry D. L., Holland K. A., Weedn V. W., Gill P. A high observed substitution rate in the human mitochondrial DNA control region. Nat Genet. 1997 Apr;15(4):363–368. doi: 10.1038/ng0497-363. [DOI] [PubMed] [Google Scholar]
  24. Phelps R. S., Chadwick R. B., Conrad M. P., Kronick M. N., Kamb A. Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening. Biotechniques. 1995 Dec;19(6):984–989. [PubMed] [Google Scholar]
  25. Piercy R., Sullivan K. M., Benson N., Gill P. The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int J Legal Med. 1993;106(2):85–90. doi: 10.1007/BF01225046. [DOI] [PubMed] [Google Scholar]
  26. Sajantila A., Lahermo P., Anttinen T., Lukka M., Sistonen P., Savontaus M. L., Aula P., Beckman L., Tranebjaerg L., Gedde-Dahl T. Genes and languages in Europe: an analysis of mitochondrial lineages. Genome Res. 1995 Aug;5(1):42–52. doi: 10.1101/gr.5.1.42. [DOI] [PubMed] [Google Scholar]
  27. Stoneking M., Hedgecock D., Higuchi R. G., Vigilant L., Erlich H. A. Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes. Am J Hum Genet. 1991 Feb;48(2):370–382. [PMC free article] [PubMed] [Google Scholar]
  28. Stoneking M. Mitochondrial DNA heteroplasmy: out of the closet. Biol Chem. 1996 Oct;377(10):603–604. [PubMed] [Google Scholar]
  29. Stoneking M., Soodyall H. Human evolution and the mitochondrial genome. Curr Opin Genet Dev. 1996 Dec;6(6):731–736. doi: 10.1016/s0959-437x(96)80028-1. [DOI] [PubMed] [Google Scholar]
  30. Sullivan K. M., Hopgood R., Gill P. Identification of human remains by amplification and automated sequencing of mitochondrial DNA. Int J Legal Med. 1992;105(2):83–86. doi: 10.1007/BF02340829. [DOI] [PubMed] [Google Scholar]
  31. Thomas A. W., Edwards A., Sherratt E. J., Majid A., Gagg J., Alcolado J. C. Molecular scanning of candidate mitochondrial tRNA genes in type 2 (non-insulin dependent) diabetes mellitus. J Med Genet. 1996 Mar;33(3):253–255. doi: 10.1136/jmg.33.3.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tully G., Sullivan K. M., Nixon P., Stones R. E., Gill P. Rapid detection of mitochondrial sequence polymorphisms using multiplex solid-phase fluorescent minisequencing. Genomics. 1996 May 15;34(1):107–113. doi: 10.1006/geno.1996.0247. [DOI] [PubMed] [Google Scholar]
  33. Wallace D. C. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science. 1992 May 1;256(5057):628–632. doi: 10.1126/science.1533953. [DOI] [PubMed] [Google Scholar]
  34. Wallace D. C., Shoffner J. M., Trounce I., Brown M. D., Ballinger S. W., Corral-Debrinski M., Horton T., Jun A. S., Lott M. T. Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim Biophys Acta. 1995 May 24;1271(1):141–151. doi: 10.1016/0925-4439(95)00021-u. [DOI] [PubMed] [Google Scholar]
  35. Watson E., Bauer K., Aman R., Weiss G., von Haeseler A., Päbo S. mtDNA sequence diversity in Africa. Am J Hum Genet. 1996 Aug;59(2):437–444. [PMC free article] [PubMed] [Google Scholar]
  36. Wilson M. R., DiZinno J. A., Polanskey D., Replogle J., Budowle B. Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med. 1995;108(2):68–74. doi: 10.1007/BF01369907. [DOI] [PubMed] [Google Scholar]
  37. Yoon K. L., Modica-Napolitano J. S., Ernst S. G., Aprille J. R. Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA. Anal Biochem. 1991 Aug 1;196(2):427–432. doi: 10.1016/0003-2697(91)90489-g. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES