Molecular Biology Freeware for Windows

A. General - below

B. Microarray - next page

C. Java programs - next page

A good places to start is Genamics SoftwareSeek. The following sites are arranged in the order that I discovered them. At some point they will be clustered by poreference:

A. DNA, RNA and genomic analysis
B. Plasmid graphic packages
C. Primer design
D. Protein analysis
E. Viewing three dimensional structures
F. Alignments
G. Phylogeny
H. Miscellaneous

I.   Graphic packages

DNA, RNA and genomic analysis:

 Gegenees is a software project for comparative analysis of whole genome sequence data and other Next Generation Sequence (NGS) data. The software can e.g. compare a large number of microbial genomes, give phylogenomic overviews and define genomic signatures unique for specified target groups. I have been using this software which permits BLASTN and TBLASTX comparisons on phage sequences in order to define relationships (Reference: Agren J et al. 2012. PLoS One. 7:e39107)

  MyRAST - It is now possible to get a fairly accurate annotation of a prokaryotic genome in about a day using this software package.  The latest Windows or Mac version of the software can be downloaded from here.  You should check out the help page - Annotating a Genome Using myRAST  and Distribution of the SEED server packages

  Tablet - Next Generation Sequence Assembly Visualization - is a lightweight, high-performance graphical viewer for next generation sequence assemblies and alignments. File format support for ACE, AFG, MAQ, SOAP2, SAM and BAM. Import GFF3 features and quickly find/highlight/display them. Search and locate reads by name across entire data sets. Entire-contig overviews, showing data layout or coverage information.

  BlastStation-Free supports megablast, blastn, blastp, and blastx searches; allows easy database creation from your FASTA or FASTQ file, which can be compressed in .gz, .Z, or .zip format. A graphical display of search results and a summary table display of search results. The latter can be exported in CSV format, while the hit sequences can be exported in FASTA format. Also available for download in Mac or PC format.

  Gene Designer- a brilliant software tools that allows one to combine building blocks such as regulatory DNA elements (promoters, ribosome-binding sites) with amino acid sequences, affinity & protease cleave tags and cloning features and codon optimize for any expression host.

CLC Free Workbench -  allows basic sequence analysis such as open reading frame determination, restriction site analysis, translation from DNA/ RNA to proteins, alignments, and tree reconstruction in a single window format.

   EMBOSS (European Molecular Biology Open Source Software Suite) can be downloaded from here.

PHIRE - this Visual Basic program performs an algorithmic string-based search on bacteriophage genome sequences, discovering and extracting blocks displaying sequence similarity, corresponding to conserved regulatory elements contained within these genomes in a systematic manner, without any prior experimental or predictive knowledge. (Reference: Lavigne, R. et al. 2004. PHIRE, a deterministic approach to reveal regulatory elements in bacteriophage genomes. Bioinformatics 20: 629-635).

MB DNA Analysis (Oleg Simakov) - MB is a free multi-functional DNA/protein analysis program. It's main advantage is that it combines all of the most widely used features needed for an advanced molecular analysis of genomic/proteomic data. Features of MB include a fast restriction analysis algorithm (included plasmid / linear DNA drawing), promoter analysis, calculation of molecular weights and chemical properties of proteins, prediction of the secondary protein structures (after Chou-Fasman). Protein analysis also includes sequence translation and codon usage table calculation. Other features: hierarchical multiple sequence alignment tool (with a feature to compare secondary structure of proteins), phylogenetic tree building, dot plot, estimation of isoelectric point for proteins, primer design. A tool for the structural analysis of alpha helices is also included in the main package.
GenePalette allows genome sequence visualization and navigation. Users can download from NCBI’s GenBank database large or small segments of genome sequence from a variety of organisms preserving the gene annotation that is associated with that sequence. Sequence elements of interest (transcription factor binding sites, etc. can be searched for and identified in the loaded sequence, and then clearly visualized within a colorful graphical representation of gene organization.

  UGene   (UniPro Bioinformatics Group, Russia) - without a doubt one of the best software packages for genome annotation (Reference: Okonechnikov K et al. 2012. Bioinformatics 28: 1166-1167).

Artemis: a DNA sequence viewer and annotation tool (Sanger Centre)

SEQtools is a program package for routine handling and analysis of DNA and protein sequences. The package includes general facilities for sequence and contig editing, restriction enzyme mapping, translation, and repeat identification.  Free for students

  DNA Club - DNA analysis software, features include remove vector sequence, find, find ORF, sequence editing, translate to protein sequence, protein sequence editing, RE Map, RE Map with translation, PCR primer selection, primer or probe evaluation etc.

  DNA for Windows is a compact, easy to use DNA analysis program, ideal for small-scale sequencing projects. 

RNAdraw: is an integrated program for RNA secondary structure calculation and analysis by Ole Matzura & Anders Wennborg  (1996) Computer Applications in the Biosciences (CABIOS) 12:  247-249

RNAstructure - RNA Secondary Structure Prediction and Analysis for Microsoft Windows.  This program includes a secondary structure prediction algorithm, a sequence editor, an integrated drawing tool, the OligoWalk program, OligoScreen, Dynalign, and a partition function calculator. (Reference: D.H. Mathews (2005) Bioinformatics 21: 2246 - 2253.)

Chromas will display and prints chromatogram files from ABI automated DNA sequencers, and Staden SCF files which the analysis programs for ALF, Li-Cor and Visible Genetics OpenGene sequencers can create.  N.B. only the older versions of the software are free.

FinchTV - Another useful tool for viewing and editing electropherograms.

  G-language Genome Analysis Environment provides a greater variety of useful genome analysis tools compared to most existing analysis software packages, and is also easily pluggable.  All of its tools are accessible as Perl modules. To get started download genome files from GenBank in *.gbk format (GenBank flat file format). 

  DNA Master - is "perhaps the world's greatest sequence editor" and analysis package. Find under "computer."

GeSTer (V. Nagaraja, Indian Institute of Science, Bangalore. India) -  is extremely useful in locating stem-loop structures, including rho-independent terminators in annotated genomes.  Since it does not run conveniently on Windows XP see how you can modify the *.gbk file so that it works.

 Staden Package - consists of a series of tools for DNA sequence preparation (pregap4), assembly (gap4), editing (gap4) and DNA/protein sequence analysis (spin). The package was originally developed at the MRC-LMB in Cambridge. It is now open source (BSD licence) and is hosted on

   Seqool - sequence analysis software designed primarily for searching biological signals in nucleic acid sequences. The sequence analysis program package provides several pattern recognition models, but it also includes the most common sequence analysis statistics, such as GC content, codon usage, etc.

  GENtle - software package for DNA and amino acid editing, database management, plasmid maps, restriction and ligation, alignments, sequencer data import, calculators, gel image display, PCR, and much more.

  RepeatAround - is designed to find “direct repeats”, “inverted repeats”, “mirror repeats” and “complementary repeats”, from 3 bp to 64 bp length, in circular genomes. It processes input files directly extracted from GenBank database or simple sequence. Outputs can be obtained in a spreadsheet containing information on the number and location of the repeats. (Reference: Goios A et al. 2006. Mitochondrion 6: 218-224.)

Plasmid graphics:

 pLOT (Jean-Marc DeKeyser, Vanderbilt University, U.S.A.)

   ApE Plasmid Editor (M. Wayne Davis, Univ. Utah, U.S.A.) highlights and draws graphic maps using feature annotations from GenBank and EMBL files; creates graphic restriction maps - linear or circular with features indicated and allows BLAST analyses along with a number of other useful features.

  pDRAW32 DNA analysis software by AcaClone software (Kjeld Olesen).  pDRAW lets you enter a DNA name and coordinates for genetic elements, such as genes, to be plotted on your DNA plots.  

 BVTech Plasmid - with this program you can draw circular or linear plasmid map with double strands or single strand. You can label the plasmid with genes and restriction sites in different colors, text, and styles. 

 Plasmid Drawing Program: Plasmidomics 0.2 (Robert Winkler, Cinvestav Unidad Irapuato, Mexico)

Primer design:

 Picky is an oligo microarray design program that identifies probes that are very unique and specific to input sequences. These calculations are based on parameters inputted by the user including optimal probe length, ideal percentage of guanine and cytosine content, target-melting temperature, salt concentration and the maximum length to which a target sequence matches any non-target sequence. (Reference: H.-H. Chou et al. (2004) Bioinformatics 20: 2893-2902).Download genome *.ffn files from GenBank for use with this program. N.B. Unfortunately these files do not include the gene names only their coordinates.

AiO (All in One) is a program for Windows, that combines typical DNA/protein features such as plasmid map drawing, finding of ORFs, translate, backtranslate, primer design and virtual cloning. AiO uses databases that allow the management of oligonucleotides, oligonucleotide-manufacturers, restriction enzymes, structural DNA and program users in a multi-user/multi-group environment. (Reference: Karreman C. (2002) Bioinformatics. 18:884-885).

- Oligo Analyzer is a simple tool to determine primer properties like Tm, GC%, primer loops, primer dimers and primer-primer compatibility. All you have to do is to paste or type primer sequence and let Oligo Analyzer to calculate all important primer properties mentioned above. Readme

- Oligo Explorer is a tool to search primers and primer pairs. The program analyzes all important primer properties like Tm, GC%, primer loops, primer dimers and etc. Readme

AnnHyb This programs features include sequence  editing with proofreading, format conversion, translation, sequence statistics, probe design & analysis. 

- MeltCalc is the ultimate thermodynamic modelling spreadsheet for Excel™ which allows you to analyze probes. See: Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches (Reference: Schütz, E., von Ahsen, N. (1999) BioTechniques 27:1218-1224).


Protein analysis:

ANTHEPROT (ANalyse THE PROTeins) is the result of biocomputing activity at the Institute of Biology and Chemistry of Proteins (Lyon, France)

  STORM - this program extracts protein sequences after ORF prediction and subsequently performs an automatic analysis for each of the proteins. This analysis consists of web-based similarity searches (BLASTp and FASTA) as well as Pfam predictions and Protparam calculations of protein physicochemical properties. The raw output for these analyses is then analysed and summarized. (Reference: Lavigne, R. et al. (2003.) Applied Bioinformatics 2: 177-179).

 Visual Evaluation and Statistics to Promote Annotation (VESPA), targeted at the integration of peptide-centric proteomics data with other forms of high-throughput, qualitative and quantitative data, such as data from Ref-SEQ analyses. At the core, VESPA integrates bottom-up proteomics data with genome level information, i.e., mapping peptides to their respective genome locations. This capability is a necessity in proteogenomics where scientists are correcting either mis-annotations or identifying new genes. The visualization allows the user to observe the location and sequence of peptides that do not match current annotations, as well as offering valuable filtering criteria such as the removal of ambiguous peptides.


Viewing three dimensional structures:

Yasara (Gregor Högenauer, Günther Koraimann, & Andreas Kungl [Univ. Graz, Austria]; & Gert Vriend [Univ. Nijmegen, the Netherlands]) is an awesome program for viewing an labeling 3-D structures.  To visual your own pdb structure right click and chose open with (Yasara).  This free program is part of a more extensive molecular modeling package.

   RasMol is software for looking at molecular structures. It is very fast: rotating a protein or DNA molecule shows its 3D structure. 

  Deep View (Swiss-PdbViewer) is an application that provides a user friendly interface allowing to analyze several proteins at the same time. The proteins can be superimposed in order to deduce structural alignments and compare their active sites or any other relevant parts. Amino acid mutations, H-bonds, angles and distances between atoms are easy to obtain thanks to the intuitive graphic and menu interface


    - Biodesigner is a molecular modeling and visualization program for personal computers which is capable of creating homologous models of proteins, evaluate, and refine the models. 

  RasTop - RasTop is a molecular visualization software adapted from the program RasMol by wrapping a user-friendly graphical interface around the "RasMol molecular engine". The software allows several molecules to be opened in the same window and several windows to be opened at the same time. Through an extended menu and a command panel, users can manipulate numerous molecules rapidly and learn about them. Work sessions are saved in script format and are fully regenerated with a simple mouse click.


ClustalX is a windows interface for the ClustalW multiple sequence alignment program. It provides an integrated environment for performing multiple sequence and profile alignments and analyzing the results.  (Reference: J.D. Thompson et al. (1997). Nucleic Acids Research 24: 4876-4882).

BioEdit is a mouse-driven, easy-to-use sequence alignment editor and sequence analysis program designed and written by Tom Hall (North Carolina State University). It also provides BLAST  capability on local databases.

  CHROMA takes your aligned multiple sequence data, annotates residues according to a consensus and displays the alignment using different font formats (text and background colours, bold and italic). The formatted annotation can be sent directly into Microsoft Word, or saved to a file or Windows Clipboard in both HTML and "Rich Text" Formats. (Reference: L. Goodstadt & C.P. Ponting. (2001) Bioinformatics 17: 845-846).

 SeaView is a graphical multiple sequence alignment editor developed by Manolo Gouy. SeaView is able to read various alignment formats (MSF, CLUSTAL, FASTA, PHYLIP, MASE). It allows ones to manually edit the alignment, and also to run DOT-PLOT or CLUSTAL programs to locally improve the alignment.

 Sequence Demarcation Tool (SDTv1.2) is a free and easy to use program that allows classification of virus sequences based on sequence pairwise identity. It takes as input a FASTA file of aligned or unaligned DNA or protein sequences and aligns every unique pair of sequences, calculates pairwise similarity scores, and displays a colour coded matrix of these scores. It also produces both a plot of these pairwise identity scores and text files containing analysis results. The identity scores are calculated as 1-(M/N) where M is the number of mismatching nucleotides and N the total number of positions along the alignment at which neither sequence has a gap character. (Reference: Muhire BM et al. (2014) PLoS ONE 9(9): e108277).


  HyPhy - intended to perform maximum likelihood analyses of genetic sequence data and equipped with tools to test various statistical hypotheses. HYPHY was designed with maximum flexibility in mind and to that end it incorporates a simple high level programming language which enables the user to tailor the analyses precisely to his or her needs. These include relative rate and ratio tests, several methods of ML based phylogeny reconstruction, bootstrapping, model selection, positive selection, molecular clock tests and many more (Reference: S.L. Kosakovsky et al.(2005) Bioinformatics 21:676-679).

TREECON - is a software package developed primarily for the construction and drawing of phylogenetic trees on the basis of evolutionary distances inferred from nucleic and amino acid sequences. It offers considerable opportunity to change the appearance of the tree. (Reference: Van de Peer, Y. & De Wachter, Y. (1994) Comput. Applic. Biosci. 10, 569-570).

TREEMAP is designed as a simple tool for visually comparing host and parasite phylogenies: you can view the host and parasite trees, interactively create reconstructions of the history of the host-parasite association, perform randomization tests of tree similarity, and (if you have branch length information such as might be obtained from DNA sequence data) compare branch lengths in the two trees. 

Treefinder (Gangolf Jobb, Statistical Genetics and Bioinformatics, University of Munich) computes phylogenetic trees from nucleotide sequences. Using the widely accepted Maximum Likelihood method, it is offering a variety of evolutionary models up to the general time reversible model with Gamma and codon position rate heterogeneity among sites. The confidence of inferred relationships may be assessed by bootstrap analysis or, alternatively, by a local rearrangement paired-sites method (LRP). Linus and Mac versions also available.

MEGA - an incredible phylogenetic analysis program. (Reference: S. Kumar et al. (2001) Bioinformatics 17: 1244-1245)..

  Tree-Puzzle (H.A. Schmidt, K. Strimmer, M. Vingron, & A. von Haeseler, Germany) constructs phylogenetic trees from molecular sequence data by maximum likelihood. It implements a fast tree search algorithm, quartet puzzling, that allows analysis of large data sets and automatically assigns estimations of support to each internal branch. TREE-PUZZLE also computes pairwise maximum likelihood distances as well as branch lengths for user specified trees. Branch lengths can be calculated under the clock- assumption. In addition, TREE-PUZZLE offers a novel method, likelihood mapping, to investigate the support of a hypothesized internal branch without computing an overall tree and to visualize the phylogenetic content of a sequence alignment.

TreeView provides a simple way to view the contents of a NEXUS, PHYLIP, or other format tree file.  This program can be coupled with an analysis package called FreeTree written by Dr. Jaroslav Flegr (Charles University, Prague, Czech Republic).

GeneTree is an experimental program for comparing gene and species trees.

  PHYLIP (the PHYLogeny Inference Package) is a package of programs for inferring phylogenies.  PHYLIP is the most widely-distributed phylogeny package, and competes with PAUP to be the one responsible for the largest number of published trees (Joe Felsenstein, University of Washington, U.S.A.).

  MrBayes is a program for Bayesian inference of phylogeny using Markov Chain Monte Carlo methods. MrBayes has a console interface and uses a modified NEXUS format for data and batch files. It handles a wide range of probabilistic models for the evolution of nucleotide and amino acid sequences, restriction sites, and standard binary data. The user can set the priors used for the parameters and search for trees under topological constraints. 

  Processor of Sequences (ProSeq) is a Windows 95 / NT program for convenient sequence alignment editing and various population-genetic analyses. PROSEQ was written by Dmitry Filatov.

PAML is a program package for phylogenetic analyses of DNA or protein sequences using maximum likelihood. It is maintained and distributed for academic use free of charge by Ziheng Yang.

  NJplot is a tree drawing program able to draw any binary tree expressed in the standard phylogenetic tree format (e.g., the format used by the PHYLIP package). NJplot is especially convenient for rooting the unrooted trees obtained from parsimony, distance or maximum likelihood tree-building methods. Written by Manolo Gouy. 

 Orthologous Average Nucleotide Identity Tool (OAT) - OAT uses OrthoANI to measure the overall similarity between two genome sequences. ANI and OrthoANI are comparable algorithms: they share the same species demarcation cut-off at 95~96% and large comparison studies have demonstrated both algorithms to produce near identical reciprocal similarities. Details of the OrthoANI algorithm is given in (Lee et al. 2015). OAT employs an easy-to-follow Graphical User Interface that allow researchers to calculate OrthoANI values between genomes of interest without unfamiliar Command Line Environments. (Reference: Lee, I. et al. (2015).  Int J Syst Evol Microbiol. 66: 1100-1103).


  SeqVerter is a sequence file format conversion utility by GeneStudio, Inc. 

DynaFit - Perform nonlinear least-squares regression on chemical or enzymatic kinetic data.

PrestoPlot - 2D plotting tool

  Xenu's Link Sleuth (TM) is a spidering software that checks Web sites for broken links. Link verification is done on "normal" links, images, frames, plug-ins, backgrounds, local image maps, style sheets, scripts and java applets. It displays a continously updated list of URLs which you can sort by different criteria. I use this program to verify if the links on Online Analysis Tools are working. 

Graphic packages:

Paint.NET is a photo and image editing tool designed for computers running Microsoft Windows XP or Windows 2000. It serves the digital imaging community as a free alternative to the standard paint application included with Windows. It brings powerful features to the desktop, a myriad of special effects, plug-in extensibility, and layer manipulation. It enhances the image editing experience for tablet owners with Windows XP Tablet Ink support. Digital photographers and artists can enhance their images with features and effects such as levels adjustment, cross-layer cloning, anti-aliased tools, motion blur, and red eye removal.

  TinyQuant is a graphical display program designed for analysis and limited manipulation of images obtained by scanning of gels or autoradiographs. Useful for integrating densities of gel bands in 16 bit greyscale (PC or Mac format ".gel" or TIFF files) or 24 bit RGB TIFF images, and for converting these to 8 bit greyscale TIFFs. 

  A Smaller GIF - Pedagoguery Software Inc. provides a variety of free software packages for both Macintosh and Windows computers.  This program reduces the size of animated GIFs without affecting their appearance in any way. 

UTHSCSA ImageTool (Dental Diagnostic Science, University of Texas Health Science Center, San Antonio, U.S.A.) - can acquire, display, edit, analyze, process, compress, save and print gray scale and color images. IT can read and write over 22 common file formats including BMP, PCX, TIF, GIF and JPEG. Image analysis functions include dimensional (distance, angle, perimeter, area) and gray scale measurements (point, line and area histogram with statistics). ImageTool supports standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection, median filtering and spatial convolutions with user-defined convolution masks.

GIMP is the GNU Image Manipulation Program. It is a freely distributed piece of software for such tasks as photo retouching, image composition and image authoring. It works on many operating systems, in many languages. The GIMP animation package, is also now available

 ACD/ChemSketch (Advanced Chemistry Development, Inc) - for drawing chemical structures and graphical images.