The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.

/pdf/clustal-w-improving-the-sensitivity-of-progressive-multiple-2na2s9sq2h.pdf

Clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

Multiple sequence alignments are fundamental to many sequence analysis methods. Most alignments are computed using the progressive alignment heuristic. These methods are starting to become a bottleneck in some analysis pipelines when faced with data sets of the size of many thousands of sequences. Some methods allow computation of larger data sets while sacrificing quality, and others produce high-quality alignments, but scale badly with the number of sequences. In this paper, we describe a new program called Clustal Omega, which can align virtually any number of protein sequences quickly and that delivers accurate alignments. The accuracy of the package on smaller test cases is similar to that of the high-quality aligners. On larger data sets, Clustal Omega outperforms other packages in terms of execution time and quality. Clustal Omega also has powerful features for adding sequences to and exploiting information in existing alignments, making use of the vast amount of precomputed information in public databases like Pfam.

/pdf/fast-scalable-generation-of-high-quality-protein-multiple-4ypo2ssntq.pdf

Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega

https://www.bioinf.uni-leipzig.de/Leere/WS1516/SeqAnalGen/T-Coffee.pdf

T-Coffee: A novel method for fast and accurate multiple sequence alignment.

Methods for alignment of protein sequences typically measure similarity by using a substitution matrix with scores for all possible exchanges of one amino acid with another. The most widely used matrices are based on the Dayhoff model of evolutionary rates. Using a different approach, we have derived substitution matrices from about 2000 blocks of aligned sequence segments characterizing more than 500 groups of related proteins. This led to marked improvements in alignments and in searches using queries from each of the groups.

https://www.pnas.org/content/pnas/89/22/10915.full.pdf

Amino acid substitution matrices from protein blocks

The shotgun sequencing strategy has been used widely in genome sequencing projects. A major phase in this strategy is to assemble short reads into long sequences. A number of DNA sequence assembly programs have been developed (Staden 1980; Peltola et al. 1984; Huang 1992; Smith et al. 1993; Gleizes and Henaut 1994; Lawrence et al. 1994; Kececioglu and Myers 1995; Sutton et al. 1995; Green 1996). The FAKII program provides a library of routines for each phase of the assembly process (Larson et al. 1996). The GAP4 program has a number of useful interactive features (Bonfield et al. 1995). The PHRAP program clips 5′ and 3′ low-quality regions of reads and uses base quality values in evaluation of overlaps and generation of contig sequences (Green 1996). TIGR Assembler has been used in a number of megabase microbial genome projects (Sutton et al. 1995). Continued development and improvement of sequence assembly programs are required to meet the challenges of the human, mouse, and maize genome projects.

We have developed the third generation of the CAP sequence assembly program (Huang 1992). The CAP3 program includes a number of improvements and new features. A capability to clip 5′ and 3′ low-quality regions of reads is included in the CAP3 program. Base quality values produced by PHRED (Ewing et al. 1998) are used in computation of overlaps between reads, construction of multiple sequence alignments of reads, and generation of consensus sequences. Efficient algorithms are employed to identify and compute overlaps between reads. Forward–reverse constraints are used to correct assembly errors and link contigs. Results of CAP3 on four BAC data sets are presented. The performance of CAP3 was compared with that of PHRAP on a number of BAC data sets. PHRAP often produces longer contigs than CAP3 whereas CAP3 often produces fewer errors in consensus sequences than PHRAP. It is easier to construct scaffolds with CAP3 than with PHRAP on low-pass data with forward–reverse constraints.

An unusual feature of CAP3 is the use of forward–reverse constraints in the construction of contigs. A forward–reverse constraint is often produced by sequencing of both ends of a subclone. A forward–reverse constraint specifies that the two reads should be on the opposite strands of the DNA molecule within a specified range of distance. By sequencing both ends of each subclone, a large number of forward–reverse constraints are produced for a cosmid or BAC data set. A difficulty with use of forward–reverse constraints in assembly is that some of the forward–reverse constraints are incorrect because of errors in lane tracking and cloning. Our strategy for dealing with this difficulty is based on the observation that a majority of the constraints are correct and wrong constraints usually occur randomly. Thus, a few unsatisfied constraints in a contig may not be sufficient to indicate an assembly error in the contig. However, if a sufficient number of constraints are all inconsistent with a join in a contig and all support an alternative join, it is likely that the current join is an error, and the alternative join should be made.

/pdf/cap3-a-dna-sequence-assembly-program-3reuvq0vvk.pdf

CAP3: A DNA Sequence Assembly Program

Multiple sequence alignment can be a useful technique for studying molecular evolution and analyzing sequence-structure relationships. Until recently, it has been impractical to apply dynamic programming, the most widely accepted method for producing pairwise alignments, to comparisons of more than three sequences. We describe the design and application of a tool for multiple alignment of amino acid sequences that implements a new algorithm that greatly reduces the computational demands of dynamic programming. This tool is able to align in reasonable time as many as eight sequences the length of an average protein.

https://www.pnas.org/content/pnas/86/12/4412.full.pdf

A tool for multiple sequence alignment

Motivated by the problem in computational biology of reconstructing the series of chromosome inversions by which one organism evolved from another, we consider the problem of computing the shortest series of reversals that transform one permutation to another. The permutations describe the order of genes on corresponding chromosomes, and areversal takes an arbitrary substring of elements, and reverses their order.

/pdf/exact-and-approximation-algorithms-for-sorting-by-reversals-4pzy2ilica.pdf

Exact and Approximation Algorithms for Sorting by Reversals, with Application to Genome Rearrangement

The trend toward very large DNA sequencing projects, such as those being undertaken as part of the Human Genome Program, necessitates the development of efficient and precise algorithms for assembling a long DNA sequence from the fragments obtained by shotgun sequencing or other methods. The sequence reconstruction problem that we take as our formulation of DNA sequence assembly is a variation of the shortest common superstring problem, complicated by the presence of sequencing errors and reverse complements of fragments. Since the simpler superstring problem is NP-hard, any efficient reconstruction procedure must resort to heuristics. In this paper, however, a four-phase approach based on rigorous design criteria is presented, and has been found to be very accurate in practice. Our method is robust in the sense that it can accommodate high sequencing error rates, and list a series of alternate solutions in the event that several appear equally good. Moreover, it uses a limited form of multiple sequence alignment to detect, and often correct, errors in the data. Our combined algorithm has successfully reconstructed nonrepetitive sequences of length 50,000 sampled at error rates of as high as 10%.

/pdf/combinatorial-algorithms-for-dna-sequence-assembly-2v3ysuc05u.pdf

Combinatorial algorithms for DNA sequence assembly

Motivation: Multiple sequence alignment is a fundamental task in bioinformatics. Current tools typically form an initial alignment by merging subalignments, and then polish this alignment by repeated splitting and merging of subalignments to obtain an improved final alignment. In general this form-and-polish strategy consists of several stages, and a profusion of methods have been tried at every stage. We carefully investigate: (1) how to utilize a new algorithm for aligning alignments that optimally solves the common subproblem of merging subalignments, and (2) what is the best choice of method for each stage to obtain the highest quality alignment.

Results: We study six stages in the form-and-polish strategy for multiple alignment: parameter choice, distance estimation, merge-tree construction, sequence-pair weighting, alignment merging, and polishing. For each stage, we consider novel approaches as well as standard ones. Interestingly, the greatest gains in alignment quality come from (i) estimating distances by a new approach using normalized alignment costs, and (ii) polishing by a new approach using 3-cuts. Experiments with a parameter-value oracle suggest large gains in quality may be possible through an input-dependent choice of alignment parameters, and we present a promising approach for building such an oracle. Combining the best approaches to each stage yields a new tool we call Opal that on benchmark alignments matches the quality of the top tools, without employing alignment consistency or hydrophobic gap penalties.

Availability:Opal, a multiple alignment tool that implements the best methods in our study, is freely available at http://opal.cs.arizona.edu

Contact: [email protected]

/pdf/multiple-alignment-by-aligning-alignments-mp3a3d76g2.pdf

Multiple alignment by aligning alignments

Software watermarking is a tool used to combat software piracy by embedding identifying information into a program. Most existing proposals for software watermarking have the shortcoming that the mark can be destroyed via fairly straightforward semantics-preserving code transformations. This paper introduces path-based watermarking, a new approach to software watermarking based on the dynamic branching behavior of programs. The advantage of this technique is that error-correcting and tamper-proofing techniques can be used to make path-based watermarks resilient against a wide variety of attacks. Experimental results, using both Java bytecode and IA-32 native code, indicate that even relatively large watermarks can be embedded into programs at modest cost.

https://www.cs.arizona.edu/~debray/Publications/pbw.pdf

John Kececioglu

Papers

A tool for multiple sequence alignment

Exact and Approximation Algorithms for Sorting by Reversals, with Application to Genome Rearrangement

Combinatorial algorithms for DNA sequence assembly

Multiple alignment by aligning alignments

Dynamic path-based software watermarking