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PolyPhred 6.18
featuring Indel Detection
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  • PolyPhred

    PolyPhred is a program that compares fluorescence-based sequences across traces obtained from different individuals to identify heterozygous sites for single nucleotide substitutions. PolyPhred is not a stand alone application. PolyPhred's functions are integrated with the use of three other programs: Phred (Brent Ewing and Phil Green), Phrap (Phil Green), and Consed (David Gordon and Phil Green). PolyPhred identifies potential heterozygotes using the base calls and peak information provided by Phred and the sequence alignments provided by Phrap. Potential heterozygotes identified by PolyPhred are marked for rapid inspection using the Consed tool.

    PolyPhred marks a column of potentially polymorphic sites in blue, with the heterozygous sites indicated in pink (see example). PolyPhred uses a ranking system to indicate how good the heterozygous sites are. The ranks range from 1 (highest) to 6 (lowest). The colored tag in the consensus sequence at the top of the column indicates the rank. The sequence traces can be examined to determine the validity of the genotype call.

    PolyPhred is not perfect, and it will occasionally miscall genotypes (example). The frequency of such mistakes depends on the sequencing chemistry used to generate the trace. This is particularly true for sequences generated with older type dye-labeled terminators and Taq-FS. To help reduce the number of miscalled sites, PolyPhred ignores regions of poor quality at the ends of sequences. In Consed, these regions appear marked in yellow (example).

    SNP detection by PolyPhred. Below is a view of a Consed window with a red rank 1 (highest) SNP tag marking the consensus position of the SNP in the traces, and genotype tags marking each of the samples (blue = homozygote, pink = heterozygote). The images to the right show traces from three of the samples: a C/C homozygote (top), a C/G heterzygote (middle) and a G/G homozygote (bottom).