Assembling Genomic DNA Sequences with PHRAP
The PHRAP assembly program provides rapid comparison, alignment, and assembly of large sets of DNA sequences. PHRAP compares sequences by searching for pairs of perfectly matching words or sequence regions that meet certain criteria. If a match is found, PHRAP then tries to extend the alignment into overlapping sections called contigs. PHRAP uses quality values produced by the PHRED basecaller to strike a balance between tolerance of discrepancies and prevention of stacking repeat sequences. The PHRAP assembly algorithm is generally used as part of the PHRED/PHRAP/Consed software suite for sequence analysis. This unit presents instructions for basic usage of the PHRAP assembler, including preparation of the input files (Support Protocols 1 and 2) and explanation of output files (Basic Protocols 1 and 2). Several command line options for changing the PHRAP assembly parameters are also discussed (Basic Protocol 3).
Keywords: PHRAP; sequence assembly; DNA
Table of Contents
- Unit Introduction
- Basic Protocol 1: Generating a PHRAP Assembly Using the Stand-Alone Version of PHRAP on an Example Data Set
- Basic Protocol 2: Generating an Assembly Using the phredPhrap Script on an Example Data Set
- Basic Protocol 3: Changing Assembly Parameters
- Support Protocol 1: Input Files
- Support Protocol 2: Read Naming Conventions
- Literature Cited
Figure 11.4.1 Example of the iy30a01.b1.qual file which is generated by PHRED and is used in the PHRAP assembly process. Note that the PHRED quality values are generally lower at the beginning or end of the sequencing read.
Figure 11.4.2 Example of the first sequence in the RICE_fasta.screen file. This is the vector masked file that is used as the input file to PHRAP for assembly. Note the Xs within the sequence that denote vector regions.
Figure 11.4.3 Example of the RICE_fasta.screen.contigs output file generated by PHRAP. Each contig sequence is listed in FASTA format.
Figure 11.4.4 Example of the beginning of the RICEphrap.out file. This file contains the standard output information from the PHRAP assembler.
Figure 11.4.5 Example of a .phd file. The beginning of the iy30a01.b1.phd.1 file generated by the phredPhrap script is shown.
Figure 11.4.6 The phredPhrap script can be customized to write the template and direction information to the bottom of the .phd file.
Figure 11.4.7 Example of a FASTA input file containing the template, chemistry, dye, and direction information in the header line.
|Ewing, B. and Green, P. 1998. Basecalling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8:186-194.|
|Ewing, B., Hillier, L., Wendl, M., and Green, P. 1998. Basecalling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8:175-185.|
|Gordon, D., Abajian, C., and Green, P. 1998. Consed: A graphical tool for sequence finishing. Genome Res. 8:195-202.|
|Gotoh, O. 1982. An improved algorithm for matching biological sequences. J. Mol. Biol. 162:705-708.|
|Smith, T.F. and Waterman, M.S. 1981. Identication of common molecular subsequences. J. Mol. Biol. 147:195-197.|
|Green, P. 1996. Documentation for PHRAP. Genome Center, University of Washington, Seattle.|