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Editing of sequences/Input before Alignment

  1. Microsoft Word
  2. Microsoft Excel
  3. Notepad++

Online Alignment tools

  1. Clustal Omega uses seeded guide trees and HMM profile-profile techniques to generate alignments between three or more sequences
  2. Kalign Fast and accurate. Suitable for larger alignments
  3. MAFFT Uses Fast Fourier Transforms. Suitable for medium-large alignments
  4. Muscle  Accurate MSA tool, especially good with proteins. Suitable for medium alignments

Standalone Alignment tools

  1. Aliview (Highly recommended) Easy GUI. Allows choosing different algorithms for aligning/realigning. Also facilitates good visualization and allows easy manual editing.
  2. Clustal Omega Latest version of Clustal. Greater Accuracy. Can align thousand of sequences in couple of hours. Influences CPU power to a significant extent. Command line/web server only. No GUI.
  3. Clustal W / Clustal X  Command-line version Clustal W and the graphical version Clustal X is available.
  4. MAFFT Only Command-line version

Automated Curation (Retrieval of conserved regions; only when absolutely needed and scientifically justified)

  1. Gblocks We recommend the online version for this tool
  2. Gapstreeze To delete aligned columns that contain a chosen percentage of gaps or other characters

Manual Curation (For deleting flanking regions or editing shifts caused by software)

  1. Genedoc
  2. Aliview 

Determination of best substitution model prior to phylogenetic analyses

  1. ProtTest
  2. SMS-Smart Model Selection

Online tools for phylogenetic analyses

  1. PhyML For maximum likelihood phylogenies. Can choose the best-fit subsitution model according to your alignment. Allows branch support analysis.
  2. It may be used for learning purposes as it is simplest of all. Runs and connects various bioinformatics programs to reconstruct a robust phylogenetic tree from a set of sequences.Has the ability to make decisions on behalf of you according to your set of sequences, which is not always recommended. Limited options of algorithms to choose from for each task, as compared to when we use their standalone versions.
  3. IQTree Web Server Can choose the best-fit subsitution model according to your alignment. Allows branch support analysis. Only for maximum likelihood trees.

Standalone softwares that allows both creating alignment and phylogenetic analysis

  1. SeaView Multiplatform, graphical user interface for multiple sequence alignment and molecular phylogeny. Easier GUI. Broad acceptance of Input formats. Allows choosing different algorithms (Muscle/Clustal) for aligning/realigning. Can compute parsimony, distance and likelihood trees. Limitation: Often leads to bugs.
  2. MEGA Allows broad number of analysis, extending beyond creating alignments and phylogenetic analyses, like Neutrality tests and Ancestral inferences. User-friendly GUI. Regulary updates and releases new version.

Standalone softwares that allows only one kind of phylogeny construction

  1. MrBayes For bayesian inference and model choice across a wide range of phylogenetic and evolutionary models. MrBayes uses Markov chain Monte Carlo (MCMC) methods to estimate the posterior distribution of model parameters. NO GUI available. Command-line interface. Widely used and accepted.
  2. PAUP* Currently, only test version is available for free downloading. The test version will expire on Jan, 2017. Command-line interface. Widely used for performing parsimonious phylogenetic reconstruction. Also distance-based and maximum likelihood-based phylogenetic reconstruction are possible.
  3. BEAST  A very advanced cross-platform program for Bayesian analysis of molecular sequences using MCMC. It is entirely orientated towards rooted, time-measured phylogenies inferred using strict or relaxed molecular clock models.

There are a number of tools that are distributed with BEAST package like:

  1. Log-Combiner - A tool that could be used to combine the logs and trees from multiple runs of BEAST/MrBAyes
  2. Tracer A program for analysing results from Bayesian MCMC programs. It allows us to access the quality, while MCMC analysis is still running, and therefore allows us to decide whether the current number of generations/states are enough and form an Effective Sample Size(ESS) or we should let the MCMC run longer?
  3. TreeAnnotator Helps in summarizing thousand of trees generated during MCMC analysis into one consensus tree. The summary information includes the posterior probabilities of the nodes in the target tree, the posterior estimates and HPD limits of the node heights and (in the case of a relaxed molecular clock model) the rates.

Visualization of Trees

  1. FigTree Highly recommended. Allows to change the rooting or changing shape of the trees. Allows to adjust scale. Especially outstanding in case of chronograms. Allows highlighting and change colors of clades etc. as per preferred presentation choices. Allows annotating tip labels. More possibilities of output format. Can not have different format within a single tip-label (This is necessary as we write organism name in Italics, but strain number in non-italics). Can export multiple graphic formats., which can be later modified in Graphic editors like Corel Draw, InkScape (Free) or Adobe photoshop.
  2. TreeView   A simple program for displaying phylogenies. Allows to choose between phylogram/cladogram. Allows to change the roots of trees and presentation (circular/rectangular). No possibility to annotate/ format tree labels. Less possibilities of output formats
  3. Dendroscope Especially good with large trees. Allows user to automatically count molecular operational taxonomic units" (MOTU) in a large tree or distinct clades. Allows find and replace. Consensus trees and rooted phylogenetic networks can be computed from a set of trees. Multiple graphic export formats.


  1. CIPRES An infra-structure for phylogenetic research. Requires user to create an account. Stores your job and run them in single/multiple remote server/s. Collection of numerous alignment and phylogeny softwares (including all which are listed above). This is highly recommended, if you have a very large data set to work with or you need to carry out multiple tasks parallely, and therefore want to keep your personal processor working normally.
  2. CLC Genomics Workbench A non-free platform that allows to analyse the sequence data generated using Next-Generating sequencing. One of the few programs that allows to assemble multiple reads generated using different sequencing techniques. It also comprises several tools for nucleotide/proteins analysis/ alignments and phylogenetic reconstruction methods.
  3. Geneious A non-free platform that allows to NGS analysis, Sequence and Chromatogram analysis, Alignment and Tree Building, Molecular cloning, Searching, Sharing and Automation of NCBI analysis.
  4. BLAST2GO It allows functional annotation of novel sequences and the analysisof annotation data. The software identifies already characterized similar sequences, and transfers its functional labels to the uncharacterized sequences. The software suite has many other functions including joined data-visualization and statistical analysis procedures. 




last updated by Komal Chenthamara, November 2017