• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Jul 15, 1997; 25(14): 2745–2751.
PMCID: PMC146817

PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing.

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.

Full Text

The Full Text of this article is available as a PDF (938K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Cooper DN, Smith BA, Cooke HJ, Niemann S, Schmidtke J. An estimate of unique DNA sequence heterozygosity in the human genome. Hum Genet. 1985;69(3):201–205. [PubMed]
  • Wallace DC. Mitochondrial DNA sequence variation in human evolution and disease. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8739–8746. [PMC free article] [PubMed]
  • Erlich HA, Bergström TF, Stoneking M, Gyllensten U. HLA sequence polymorphism and the origin of humans. Science. 1996 Nov 29;274(5292):1552–1554. [PubMed]
  • Trivier E, De Cesare D, Jacquot S, Pannetier S, Zackai E, Young I, Mandel JL, Sassone-Corsi P, Hanauer A. Mutations in the kinase Rsk-2 associated with Coffin-Lowry syndrome. Nature. 1996 Dec 12;384(6609):567–570. [PubMed]
  • de Kok YJ, van der Maarel SM, Bitner-Glindzicz M, Huber I, Monaco AP, Malcolm S, Pembrey ME, Ropers HH, Cremers FP. Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science. 1995 Feb 3;267(5198):685–688. [PubMed]
  • 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):118–129. [PubMed]
  • Shattuck-Eidens D, McClure M, Simard J, Labrie F, Narod S, Couch F, Hoskins K, Weber B, Castilla L, Erdos M, et al. 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]
  • Santamaria P, Boyce-Jacino MT, Lindstrom AL, Barbosa JJ, Faras AJ, Rich SS. HLA class II "typing": direct sequencing of DRB, DQB, and DQA genes. Hum Immunol. 1992 Feb;33(2):69–81. [PubMed]
  • Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle B. Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med. 1995;108(2):68–74. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Sheffield VC, Cox DR, Lerman LS, Myers RM. 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. [PMC free article] [PubMed]
  • Cotton RG, Rodrigues NR, Campbell RD. 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. [PMC free article] [PubMed]
  • Myers RM, 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. [PubMed]
  • Youil R, Kemper BW, Cotton RG. 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. [PMC free article] [PubMed]
  • Glavac D, Dean M. Applications of heteroduplex analysis for mutation detection in disease genes. Hum Mutat. 1995;6(4):281–287. [PubMed]
  • 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. [PubMed]
  • Hacia JG, Brody LC, Chee MS, Fodor SP, Collins FS. Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis. Nat Genet. 1996 Dec;14(4):441–447. [PubMed]
  • Chee M, Yang R, Hubbell E, Berno A, Huang XC, Stern D, Winkler J, Lockhart DJ, Morris MS, Fodor SP. Accessing genetic information with high-density DNA arrays. Science. 1996 Oct 25;274(5287):610–614. [PubMed]
  • Gibbs RA, Nguyen PN, McBride LJ, Koepf SM, Caskey CT. 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. [PMC free article] [PubMed]
  • Leren TP, Rødningen OK, 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]
  • Kwok PY, Carlson C, Yager TD, Ankener W, Nickerson DA. Comparative analysis of human DNA variations by fluorescence-based sequencing of PCR products. Genomics. 1994 Sep 1;23(1):138–144. [PubMed]
  • Phelps RS, Chadwick RB, Conrad MP, Kronick MN, Kamb A. Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening. Biotechniques. 1995 Dec;19(6):984–989. [PubMed]
  • Versluis LF, Rozemuller E, Tonks S, Marsh SG, Bouwens AG, Bodmer JG, Tilanus MG. 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. [PubMed]
  • 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. [PubMed]
  • Parker LT, Zakeri H, Deng Q, Spurgeon S, Kwok PY, Nickerson DA. AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns. Biotechniques. 1996 Oct;21(4):694–699. [PubMed]
  • Nickerson DA, 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. [PubMed]
  • Boysen C, Carlson C, Hood E, Hood L, Nickerson DA. Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products. Immunogenetics. 1996;44(2):121–127. [PubMed]
  • Tobe VO, Taylor SL, Nickerson DA. 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. [PMC free article] [PubMed]
  • Ju J, Ruan C, Fuller CW, Glazer AN, Mathies RA. 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. [PMC free article] [PubMed]
  • Metzker ML, Lu J, Gibbs RA. Electrophoretically uniform fluorescent dyes for automated DNA sequencing. Science. 1996 Mar 8;271(5254):1420–1422. [PubMed]
  • Kwok PY, Deng Q, Zakeri H, Taylor SL, Nickerson DA. Increasing the information content of STS-based genome maps: identifying polymorphisms in mapped STSs. Genomics. 1996 Jan 1;31(1):123–126. [PubMed]
  • Grompe M. The rapid detection of unknown mutations in nucleic acids. Nat Genet. 1993 Oct;5(2):111–117. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...