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Phrap is a widely used program for DNA
sequence assembly In bioinformatics, sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology might not be able to 'read' whole genomes in one ...
. It is part of the Phred-Phrap-
Consed Consed is a program for viewing, editing, and finishing DNA sequence assemblies. Originally developed for sequence assemblies created with phrap, recent versions also support other sequence assembly programs like Newbler. History Consed was ...
package.


History

Phrap was originally developed by Prof. Phil Green for the assembly of
cosmid A cosmid is a type of hybrid plasmid that contains a Lambda phage ''cos'' sequence. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in ...
s in large-scale cosmid shotgun sequencing within the
Human Genome Project The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a ...
. Phrap has been widely used for many different sequence assembly projects, including bacterial genome assemblies and EST assemblies. Phrap was written as a command line program for easy integration into automated data workflows in genome sequencing centers. For users who want to use Phrap from a graphical interface, the commercial programs
MacVector MacVector is a commercial sequence analysis application for Apple Macintosh computers running Mac OS X. It is intended to be used by Molecular biology, molecular biologists to help analyze, design, research and document their experiments in the la ...
(for
Mac OS X macOS (; previously OS X and originally Mac OS X) is a Unix operating system developed and marketed by Apple Inc. since 2001. It is the primary operating system for Apple's Mac (computer), Mac computers. Within the market of ...
only) and
CodonCode Aligner CodonCode Aligner is a commercial application for DNA sequence assembly, sequence alignment, and editing on Mac OS X and Windows. Features Features include chromatogram editing, end clipping, and vector trimming, sequence assembly and contig e ...
(for
Mac OS X macOS (; previously OS X and originally Mac OS X) is a Unix operating system developed and marketed by Apple Inc. since 2001. It is the primary operating system for Apple's Mac (computer), Mac computers. Within the market of ...
and
Microsoft Windows Windows is a group of several proprietary graphical operating system families developed and marketed by Microsoft. Each family caters to a certain sector of the computing industry. For example, Windows NT for consumers, Windows Server for serv ...
) are available.


Methods

A detailed (albeit partially outdated) description of the Phrap algorithms can be found in th
Phrap documentation
A recurring thread within the Phrap algorithms is the use of
Phred quality score A Phred quality score is a measure of the quality of the identification of the nucleobases generated by automated DNA sequencing. It was originally developed for the computer program Phred (software), Phred to help in the automation of DNA sequenci ...
s. Phrap used quality scores to mitigate a problem that other assembly programs had struggled with at the beginning of the
Human Genome Project The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a ...
: correctly assembling frequent imperfect repeats, in particular
Alu sequence An Alu element is a short stretch of DNA originally characterized by the action of the ''Arthrobacter luteus (Alu)'' restriction endonuclease. ''Alu'' elements are the most abundant transposable elements, containing over one million copies disp ...
s. Phrap uses quality scores to tell if any observed differences in repeated regions are likely to be due to random ambiguities in the sequencing process, or more likely to be due to the sequences being from different copies of the Alu repeat. Typically, Phrap had no problems differentiating between the different Alu copies in a cosmid, and to correctly assemble the cosmids (or, later, BACs). The logic is simple: a base call with a high probability of being correct should never be aligned with another high quality but different base. However, Phrap does not rule out such alignments entirely, and the cross_match alignment gap and alignment penalties used while looking for local alignments are not always optimal for typical sequencing errors and a search for overlapping (contiguous) sequences. (Affine gaps are helpful for homology searches but not usually for sequencing error alignment). Phrap attempts to classify chimeras, vector sequences and low quality end regions all in a single alignment and will sometimes make mistakes. Furthermore, Phrap has more than one round of assembly building internally and later rounds are less stringent - Greedy algorithm. These design choices were helpful in the 1990s when the program was originally written (at
Washington University in St. Louis Washington University in St. Louis (WashU or WUSTL) is a private research university with its main campus in St. Louis County, and Clayton, Missouri. Founded in 1853, the university is named after George Washington. Washington University is r ...
) but are less so now. Phrap appears error prone in comparison with newer assemblers like Euler and cannot use mate-pair information directly to guide assembly and assemble past perfect repeats. Phrap is not free software so it has not been extended and enhanced like less restricted open-source software
Sequence assembly In bioinformatics, sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology might not be able to 'read' whole genomes in one ...
.


Quality based consensus sequences

Another use of
Phred quality score A Phred quality score is a measure of the quality of the identification of the nucleobases generated by automated DNA sequencing. It was originally developed for the computer program Phred (software), Phred to help in the automation of DNA sequenci ...
s by Phrap that contributed to the program's success was the determination of consensus sequences using sequence qualities. In effect, Phrap automated a step that was a major bottleneck in the early phases of the
Human Genome Project The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a ...
: to determine the correct consensus sequence at all positions where the assembled sequences had discrepant bases. This approach had been suggested by Bonfield and Staden in 1995, and was implemented and further optimized in Phrap. Basically, at any consensus position with discrepant bases, Phrap examines the quality scores of the aligned sequences to find the highest quality sequence. In the process, Phrap takes confirmation of local sequence by other reads into account, after considering direction and sequencing chemistry. The mathematics of this approach were rather simple, since
Phred quality score A Phred quality score is a measure of the quality of the identification of the nucleobases generated by automated DNA sequencing. It was originally developed for the computer program Phred (software), Phred to help in the automation of DNA sequenci ...
s are logarithmically linked to error probabilities. This means that the quality scores of confirming reads can simply be added, as long as the error distributions are sufficiently independent. To satisfy this independence criterion, reads must typically be in different direction, since peak patterns that cause base calling errors are often identical when a region is sequenced several times in the same direction. If a consensus base is covered by both high-quality sequence and (discrepant) low-quality sequence, Phrap's selection of the higher quality sequence will in most cases be correct. Phrap then assigns the confirmed base quality to the consensus sequence base. This makes it easy to (a) find consensus regions that are not covered by high quality sequence (which will also have low quality), and (b) to quickly calculate a reasonably accurate estimate of the error rate of the consensus sequence. This information can then be used to direct finishing efforts, for example re-sequencing of problem regions. The combination of accurate, base-specific quality scores and a quality-based consensus sequence was a critical element in the success of the
Human Genome Project The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a ...
. Phred and Phrap, and similar programs who picked up on the ideas pioneered by these two programs, enabled the assembly of large parts of the human genome (and many other genomes) at an accuracy that was substantially higher (less than 1 error in 10,000 bases) than the typical accuracy of carefully hand-edited sequences that had been submitted to the
GenBank The GenBank sequence database is an open access, annotated collection of all publicly available nucleotide sequences and their protein translations. It is produced and maintained by the National Center for Biotechnology Information (NCBI; a part ...
database before.Krawetz SA (1989): Sequence errors described in GenBank: a means to determine the accuracy of DNA sequence interpretation. Nucleic Acids Res. 1989 May 25;17(10):3951-7


References


External links


Phrap homepage


Other Software

* Phred *
Consed Consed is a program for viewing, editing, and finishing DNA sequence assemblies. Originally developed for sequence assemblies created with phrap, recent versions also support other sequence assembly programs like Newbler. History Consed was ...
* DNA Baser Command Line Tool Bioinformatics software Computational science