We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the logexpectation score, and refinement using treedependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

MUSCLE: multiple sequence alignment with high accuracy and high throughput

We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.

https://www.science-open.com/document_file/00d79e6c-98e4-425c-9fd1-3a85d5fdefbc/PubMedCentral/00d79e6c-98e4-425c-9fd1-3a85d5fdefbc.pdf

MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability

Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or “transition” type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or “transversion” type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = — (1/2) ln {(1 — 2P — Q) }. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'S = — (1/2) ln (1 — 2P — Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.

A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.

Summary: The Clustal W and Clustal X multiple sequence alignment programs have been completely rewritten in C++. This will facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems.

Availability: The programs can be run on-line from the EBI web server: http://www.ebi.ac.uk/tools/clustalw2. The source code and executables for Windows, Linux and Macintosh computers are available from the EBI ftp site ftp://ftp.ebi.ac.uk/pub/software/clustalw2/

Contact: clustalw@ucd.ie

Clustal W and Clustal X version 2.0

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

A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing methods. MAFFT includes two novel techniques. (i) Homologous regions are rapidly identified by the fast Fourier transform (FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length. Two different heuristics, the progressive method (FFT-NS-2) and the iterative refinement method (FFT-NS-i), are implemented in MAFFT. The performances of FFT-NS-2 and FFT-NS-i were compared with other methods by computer simulations and benchmark tests; the CPU time of FFT-NS-2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT-NS-i is over 100 times faster than T-COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.

/pdf/mafft-a-novel-method-for-rapid-multiple-sequence-alignment-485azc27cn.pdf

MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform

A method for estimating the evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences is presented. This method is applied to genes of oX174 and G4 genomes, histone genes andβ-globin genes, for which homologous nucleotide sequences are available for comparison to be made. It is shown that the rates of synonymous substitutions are quite uniform among the non-overlapping genes of oX174 and G4 and among histone genes H4, H2B, H3 and H2A. A comparison between oX174 and G4 reveals that, in the overlapping segments of the A-gene, the rate of synonymous substitution is reduced more significantly than the rate of amino acid substitution relative to the corresponding rate in the nonoverlapping segment. It is also suggested that, in the coding regions surrounding the splicing points of intervening sequences ofβ-globin genes, there exist rigid secondary structures. It is in only these regions that theβ-globin genes show the slowing down of evolutionary rates of both synonymous and amino acid substitutions in the primate line.

Molecular Evolution of mRNA: A Method for Estimating Evolutionary Rates of Synonymous and Amino Acid Substitutions from Homologous Nucleotide Sequences and Its Application

The phylogenetic position of turtles is a currently controversial issue. Recent molecular studies rejected a traditional view that turtles are basal living reptiles (Hedges, S. B., and L. L. Poling. 1999. A molecular phylogeny. Science 83:998-1001; Kumazawa, Y., and M. Nishida. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink, statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16:784-792). Instead, these studies grouped turtles with birds and crocodiles. The relationship among turtles, birds, and crocodiles remained unclear to date. To resolve this issue, we have cloned and sequenced two nuclear genes encoding the catalytic subunit of DNA polymerase alpha and glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase from amniotes and an amphibian. The amino acid sequences of these proteins were subjected to a phylogenetic analysis based on the maximum likelihood method. The resulting tree showed that turtles are the sister group to a monophyletic cluster of archosaurs (birds and crocodiles). All other possible tree topologies were significantly rejected.

Sister Group Relationship of Turtles to the Bird-Crocodilian Clade Revealed by Nuclear DNA–Coded Proteins

Phylogenetic analyses of jawed vertebrates based on mitochondrial sequences often result in confusing inferences which are obviously inconsistent with generally accepted trees. In particular, in a hypothesis by Rasmussen and Arnason based on mitochondrial trees, cartilaginous fishes have a terminal position in a paraphyletic cluster of bony fishes. No previous analysis based on nuclear DNA-coded genes could significantly reject the mitochondrial trees of jawed vertebrates. We have cloned and sequenced seven nuclear DNA-coded genes from 13 vertebrate species. These sequences, together with sequences available from databases including 13 jawed vertebrates from eight major groups (cartilaginous fishes, bichir, chondrosteans, gar, bowfin, teleost fishes, lungfishes and tetrapods) and an outgroup (a cyclostome and a lancelet), have been subjected to phylogenetic analyses based on the maximum likelihood method. Cartilaginous fishes have been inferred to be basal to other jawed vertebrates, which is consistent with the generally accepted view. The minimum log-likelihood difference between the maximum likelihood tree and trees not supporting the basal position of cartilaginous fishes is 18.3 ± 13.1. The hypothesis by Rasmussen and Arnason has been significantly rejected with the minimum log-likelihood difference of 123 ± 23.3. Our tree has also shown that living holosteans, comprising bowfin and gar, form a monophyletic group which is the sister group to teleost fishes. This is consistent with a formerly prevalent view of vertebrate classification, although inconsistent with both of the current morphology-based and mitochondrial sequence-based trees. Furthermore, the bichir has been shown to be the basal ray-finned fish. Tetrapods and lungfish have formed a monophyletic cluster in the tree inferred from the concatenated alignment, being consistent with the currently prevalent view. It also remains possible that tetrapods are more closely related to ray-finned fishes than to lungfishes.

/pdf/basal-jawed-vertebrate-phylogeny-inferred-from-multiple-2rw9pgyf1p.pdf

Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes

A heuristic approach to search for the maximum-likelihood (ML) phylogenetic tree based on a genetic algorithm (GA) has been developed. It outputs the best tree as well as multiple alternative trees that are not significantly worse than the best one on the basis of the likelihood criterion. These near-optimum trees are subjected to further statistical tests. This approach enables ones to infer phylogenetic trees of over 20 taxa taking account of the rate heterogeneity among sites on practical time scales on a PC cluster. Computer simulations were conducted to compare the efficiency of the present approach with that of several likelihood-based methods and distance-based methods, using amino acid sequence data of relatively large (5-24) taxa. The superiority of the ML method over distance-based methods increases as the condition of simulations becomes more realistic (an incorrect model is assumed or many taxa are involved). This approach was applied to the inference of the universal tree based on the concatenated amino acid sequences of vertically descendent genes that are shared among all genomes whose complete sequences have been reported. The inferred tree strongly supports that Archaea is paraphyletic and Eukarya is specifically related to Crenarchaeota. Apart from the paraphyly of Archaea and some minor disagreements, the universal tree based on these genes is largely consistent with the universal tree based on SSU rRNA.

Takashi Miyata

Papers

MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform

Molecular Evolution of mRNA: A Method for Estimating Evolutionary Rates of Synonymous and Amino Acid Substitutions from Homologous Nucleotide Sequences and Its Application

Sister Group Relationship of Turtles to the Bird-Crocodilian Clade Revealed by Nuclear DNA–Coded Proteins

Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes

Genetic algorithm-based maximum-likelihood analysis for molecular phylogeny.