PolyPhred Analysis Software for Mutation Detection from Fluorescence‐Based Sequence Data
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- Abstract
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Abstract
The ability to search for genetic variants that may be related to human disease is one of the most exciting consequences of the availability of the sequence of the human genome. Large cohorts of individuals exhibiting certain phenotypes can be studied and candidate genes resequenced. However, the challenge of analyzing sequence data from many individuals with accuracy, speed, and economy is great. This unit describes one set of software tools: Phred, Phrap, PolyPhred, and Consed. Coverage includes the advantages and disadvantages of these analysis tools, details for obtaining and using the software, and the results one may expect. The software is being continually updated to permit further automation of mutation analysis. Currently, however, at least some manual review is required if one wishes to identify 100% of the variants in a sample set. Curr. Protoc. Hum. Genet. 59:7.16.1?7.16.21. © 2008 by John Wiley & Sons, Inc.
Keywords: DNA sequencing; mutation identification; SNPs; indels; sequence traces; Consed; Phred; Phrap; PolyPhred
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Strategic Planning
- Basic Protocol 1: Using PolyPhred for Sequence Analysis and Mutation Detection
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1: Using PolyPhred for Sequence Analysis and Mutation Detection
Materials
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Figure 7.16.1 Consed Main Window. Drop‐down menus applying to the whole project are on top, then a box listing the .ace file being reviewed. Below the .ace file are several action buttons and then the Contig List and Read List. Two boxes are available for performing searches based on read name, and two action buttons that will either Show a contig or read highlighted in one of the lists or Close All Windows. The Quit Consed button (on the upper right) will exit the program after prompting for saving your changes. The Help button (on the top right) will give you full documentation. View Image -
Figure 7.16.2 Aligned Reads Window, PolyPhred v.5.04. Read names are on the left, and arrows indicate the direction of each read. Here, forward is on the top and reverse on the bottom. The quality of bases is indicated by the shading, from dark gray to white. The colored red bar on the pseudo sequence is a coding sequence tag added by the user, while the column of blue tags is applied by PolyPhred to indicate a position where a polymorphism is identified. The red tag on the consensus means this polymorphism is a polyPhredRank1 tag, with a quality score of 99, and the pink tags indicate heterozygous bases. View Image -
Figure 7.16.3 (A ) Aligned reads window after changing view from “strand” to “alpha” in the Options list. This view facilitates review by placing forward and reverse reads next to one another. The red tag at the top indicates the polymorphism is Rank1, with a quality score of 99. Two individuals are shown with heterozygous tags (pink) and two with homozygous tags (blue). (B ) The chromatograms showing heterozygous and homozygous individuals in both directions. See fuschia arrows in panel A for these data (021.F at the top, then 021.R, 019.F, and 019.R). View Image -
Figure 7.16.4 Aligned Reads window showing four individuals with heterozygous single‐base deletions. The characteristic pattern for indels is clear sequence to the point of the deletion or insertion, then an immediate loss of good sequence. The arrows indicate the direction of the reads. PolyPhred v.5.04 does not tag the indels, so manual review is required to identify them. View Image -
Figure 7.16.5 Traces showing a single‐base deletion. The top panel is the forward trace of a normal individual, the middle panel shows the forward sequence of an individual with a deletion, and the bottom panel shows the reverse sequence of the same individual. View Image
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Literature Cited
| Literature Cited | |
| Bhangale, T.R., Stephens, M., and Nickerson, D.A. 2006. Automating resequencing‐based detection of insertion‐deletion polymorphisms. Nat. Genet. 38:1457‐1462. | |
| Ewing, B. and Green, P. 1998. Base‐calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8:186‐194. | |
| Ewing, B., Hillier, L., Wendl, M., Green, P. 1998. Basecalling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8:175‐185. | |
| Gordon, D. 2004. Viewing and editing assembled sequences using consed. Curr. Protoc. Bioinformatics 11.2.1‐11.2.43. | |
| Gordon, D., Abajian, C., and Green, P. 1998. Consed: A graphical tool for sequence finishing. Genome Res. 8:195‐202. | |
| Nickerson, D.A., Tobe, V.O., and Taylor, S.L. 1997. PolyPhred: Automating the detection and genotyping of single nucleotide substitutions using fluorescence‐based resequencing. Nucleic Acids Res. 25:2745‐2751. | |
| Staden, R. 1994. Staden: Comparing sequences. Methods Mol. Biol. 25:155‐170. | |
| Stephens, M., Sloan, J.S., Robertson, P.D., Scheet, P., and Nickerson, D.A. 2006. Automating sequence‐based detection and genotyping of SNPs from diploid samples. Nat. Genet. 38:375‐381. |
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