PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing (original) (raw)

Abstract

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.

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Selected References

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  1. Boysen C., Carlson C., Hood E., Hood L., Nickerson D. A. Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products. Immunogenetics. 1996;44(2):121–127. [PubMed] [Google Scholar]
  2. 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]
  3. Cooper D. N., Smith B. A., Cooke H. J., Niemann S., Schmidtke J. An estimate of unique DNA sequence heterozygosity in the human genome. Hum Genet. 1985;69(3):201–205. doi: 10.1007/BF00293024. [DOI] [PubMed] [Google Scholar]
  4. Cotton R. G., Rodrigues N. R., Campbell R. D. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4397–4401. doi: 10.1073/pnas.85.12.4397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Erlich H. A., Bergström T. F., Stoneking M., Gyllensten U. HLA sequence polymorphism and the origin of humans. Science. 1996 Nov 29;274(5292):1552–1554. [PubMed] [Google Scholar]
  6. Gibbs R. A., Nguyen P. N., McBride L. J., Koepf S. M., Caskey C. T. Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1919–1923. doi: 10.1073/pnas.86.6.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glavac D., Dean M. Applications of heteroduplex analysis for mutation detection in disease genes. Hum Mutat. 1995;6(4):281–287. doi: 10.1002/humu.1380060402. [DOI] [PubMed] [Google Scholar]
  8. Grompe M. The rapid detection of unknown mutations in nucleic acids. Nat Genet. 1993 Oct;5(2):111–117. doi: 10.1038/ng1093-111. [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. Hattori M., Shibata A., Yoshioka K., Sakaki Y. Orphan peak analysis: a novel method for detection of point mutations using an automated fluorescence DNA sequencer. Genomics. 1993 Feb;15(2):415–417. doi: 10.1006/geno.1993.1077. [DOI] [PubMed] [Google Scholar]
  11. Hedrum A., Pontén F., Ren Z., Lundeberg J., Pontén J., Uhlén M. Sequence-based analysis of the human p53 gene based on microdissection of tumor biopsy samples. Biotechniques. 1994 Jul;17(1):118-9, 122-4, 126-9. [PubMed] [Google Scholar]
  12. Ju J., Ruan C., Fuller C. W., Glazer A. N., Mathies R. A. Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4347–4351. doi: 10.1073/pnas.92.10.4347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Kwok P. Y., Deng Q., Zakeri H., Taylor S. L., Nickerson D. A. Increasing the information content of STS-based genome maps: identifying polymorphisms in mapped STSs. Genomics. 1996 Jan 1;31(1):123–126. doi: 10.1006/geno.1996.0019. [DOI] [PubMed] [Google Scholar]
  15. Leren T. P., Rødningen O. K., Røsby O., Solberg K., Berg K. Screening for point mutations by semi-automated DNA sequencing using sequenase and magnetic beads. Biotechniques. 1993 Apr;14(4):618–623. [PubMed] [Google Scholar]
  16. 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]
  17. Myers R. M., Larin Z., Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. 1985 Dec 13;230(4731):1242–1246. doi: 10.1126/science.4071043. [DOI] [PubMed] [Google Scholar]
  18. Nickerson D. A., Whitehurst C., Boysen C., Charmley P., Kaiser R., Hood L. Identification of clusters of biallelic polymorphic sequence-tagged sites (pSTSs) that generate highly informative and automatable markers for genetic linkage mapping. Genomics. 1992 Feb;12(2):377–387. doi: 10.1016/0888-7543(92)90388-9. [DOI] [PubMed] [Google Scholar]
  19. Orita M., Suzuki Y., Sekiya T., Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics. 1989 Nov;5(4):874–879. doi: 10.1016/0888-7543(89)90129-8. [DOI] [PubMed] [Google Scholar]
  20. Parker L. T., Zakeri H., Deng Q., Spurgeon S., Kwok P. Y., Nickerson D. A. AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns. Biotechniques. 1996 Oct;21(4):694–699. doi: 10.2144/96214rr02. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  23. Santamaria P., Boyce-Jacino M. T., Lindstrom A. L., Barbosa J. J., Faras A. J., Rich S. S. HLA class II "typing": direct sequencing of DRB, DQB, and DQA genes. Hum Immunol. 1992 Feb;33(2):69–81. doi: 10.1016/0198-8859(92)90056-s. [DOI] [PubMed] [Google Scholar]
  24. Shattuck-Eidens D., McClure M., Simard J., Labrie F., Narod S., Couch F., Hoskins K., Weber B., Castilla L., Erdos M. A collaborative survey of 80 mutations in the BRCA1 breast and ovarian cancer susceptibility gene. Implications for presymptomatic testing and screening. JAMA. 1995 Feb 15;273(7):535–541. [PubMed] [Google Scholar]
  25. Sheffield V. C., Cox D. R., Lerman L. S., Myers R. M. Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A. 1989 Jan;86(1):232–236. doi: 10.1073/pnas.86.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tobe V. O., Taylor S. L., Nickerson D. A. Single-well genotyping of diallelic sequence variations by a two-color ELISA-based oligonucleotide ligation assay. Nucleic Acids Res. 1996 Oct 1;24(19):3728–3732. doi: 10.1093/nar/24.19.3728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Trivier E., De Cesare D., Jacquot S., Pannetier S., Zackai E., Young I., Mandel J. L., Sassone-Corsi P., Hanauer A. Mutations in the kinase Rsk-2 associated with Coffin-Lowry syndrome. Nature. 1996 Dec 12;384(6609):567–570. doi: 10.1038/384567a0. [DOI] [PubMed] [Google Scholar]
  28. Versluis L. F., Rozemuller E., Tonks S., Marsh S. G., Bouwens A. G., Bodmer J. G., Tilanus M. G. High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2. Hum Immunol. 1993 Dec;38(4):277–283. doi: 10.1016/0198-8859(93)90555-f. [DOI] [PubMed] [Google Scholar]
  29. Wallace D. C. Mitochondrial DNA sequence variation in human evolution and disease. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8739–8746. doi: 10.1073/pnas.91.19.8739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Youil R., Kemper B. W., Cotton R. G. Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):87–91. doi: 10.1073/pnas.92.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. de Kok Y. J., van der Maarel S. M., Bitner-Glindzicz M., Huber I., Monaco A. P., Malcolm S., Pembrey M. E., Ropers H. H., Cremers F. P. Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science. 1995 Feb 3;267(5198):685–688. doi: 10.1126/science.7839145. [DOI] [PubMed] [Google Scholar]