Software review. Vector NTI, a balanced all-in-one sequence analysis suite

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1 Vector NTI, a balanced all-in-one sequence analysis suite Keywords: sequence analysis, software package, database, virtual cloning, sequence assembly Abstract Vector NTI is a well-balanced desktop application integrated for molecular sequence analysis and biological data management. It has a centralised database and five application modules: Vector NTI, AlignX, BioAnnotator, ContigExpress and GenomBench. In this review, the features and functions available in this software are examined. These include database management, primer design, virtual cloning, alignments, sequence assembly, 3D molecular viewer and internet tools. Some problems encountered when using this software are also discussed. It is hoped that this review will introduce this software to more molecular biologists so they can make better-informed decisions when choosing computational tools to facilitate their everyday laboratory work. This tool can save time and enhance analysis but it requires some learning on the user s part and there are some issues that need to be addressed by the developer. INTRODUCTION During the last two decades a variety of computer programs (or packages) have been developed for molecular sequence analysis. Most of them are freeware, which greatly benefits researchers with limited budgets and bioinformaticians who want to access source codes to modify and/or integrate them into other tools. However, free software to some extent lacks integration with other functions, and many do not have a userfriendly graphic interface. 1 Commercial software packages, on the other hand, usually incorporate many functions, have easy-to-use user interfaces, and offer user support. Many bench scientists thus prefer to use a single all-in-one commercial software package despite their much higher costs. 2 Vector NTI is an integrated desktop application for molecular biologists. It was recognised as the best 2003 sequence analysis software by BioScience Technology readers. 3 Although Vector NTI has been reviewed previously, 4,5 these reviews were based on the old versions. At least two more versions have been released since then, with current versions being Vector NTI Advance 9.0 for Windows and Vector NTI Suite 7.1 for Macintosh platforms. In addition, InforMax was acquired by Invitrogen 6 in late 2002, and Invitrogen has added some new features into Vector NTI (eg Gateway Cloning). The Vector NTI User s Manual is helpful. However, its huge volume (eg 694 pages for Vector NTI Advance 9.0) to some extent holds back molecular biologists intention to use this package. The authors of this review have been offering a bioinformatics tutorial course and Vector NTI workshops to faculty, staff and students at the University of Nebraska-Lincoln. Many who took our courses recognise Vector NTI as a comprehensive and user-friendly sequence analysis package. In this review, useful features as well as some problems 378 & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER 2004

2 found in Vector NTI are discussed for both Windows and Macintosh platforms. THE SOFTWARE Overview Vector NTI Advance (or Vector NTI Suite) consists of a central database and five application modules. The database provides an interface to access all application modules. The following modules are available: Vector NTI for sequence creation, mapping and analysis; AlignX for multiple sequence alignment; BioAnnotator for nucleotide and protein sequence analysis; ContigExpress for sequence assembly and sequencing project management; and GenomBench for analysis and annotation of reference genomic DNA sequences. In Vector NTI Suite for Macintosh, GenomBench does not exist and BioAnnotator is called BioPlot. Vector NTI installation Vector NTI software can be downloaded from the Invitrogen web site. 6 Free registration is required for downloading. The minimal system requirements for Vector NTI Advance 9 are Microsoft Windows 98 SE or above, 500 Mb HD space, 128 Mb RAM and Microsoft Installer Version 2. The minimal requirements for Vector NTI Suite 7.1 for Macintosh are Mac OS X v or above, Power Mac G3 or G4, 450 Mb HD space, and 256 Mb RAM. A single Vector NTI database can be installed and shared by multiple users. Each user can also create a unique database in a separate location. Three types of licences are available: trial, static and dynamic. A trial licence gives the user a fully functional copy of the software free for 20 days. Vector NTI database Vector NTI uses the database and graphic user interface, namely Database Explorer (Vector NTI Explorer for Macintosh), to manage data (Figure 1). This database concept is the first thing the first-time Figure 1: Vector NTI Database Explorer window & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER

3 users need to get used to. It has both advantage and disadvantage. Using the database concept is good for a large-scale data management. On the other hand, if you want to do a small job quickly, you will be frustrated until you become familiar with the way Vector NTI wants you to manage your data. Before you can start creating your own sequence file and start any analysis, at a minimum, you need to understand some terminologies such as main database, sub-bases and objects. The database consists of seven types of objects: DNA/RNA molecules, protein molecules, enzymes, oligos, gel markers, citations and BLAST results. Vector NTI Advance has also analysis results object. The user creates new sub-bases to archive records on, for example, molecules created in the laboratory, enzymes stored in a laboratory freezer, or literature collected for a research project. The Explorer makes it relatively simple to manage and manipulate various kinds of data. With the Explorer, the user can create and edit molecules, perform database searches, import and export data, create archives of molecules and analysis results to share with other users. Vector NTI takes into consideration general security by providing database backup and restoring functions. On the other hand, to avoid quickly filling up the disk space and cluttering the work space, it is suggested that the user cleans up the database regularly. When you start Vector NTI for the first time after installation, Vector NTI creates an empty database. The user can choose to import an initial set of database objects from Vector NTI archives that include a large number of samples of DNA molecules, proteins, enzymes, oligos and gel markers. Molecule Display The Molecule Display window, also called Molecule Viewer, is the primary interface to display and manipulate DNA/RNA and protein molecules in the module Vector NTI (Figure 2). It has a three-pane format: Text pane containing molecule information (eg general description, feature map, original author) in hierarchical folders, Graphics pane that graphically displays the data in the Figure 2: Molecule Display window for a DNA molecule, PBR & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER 2004

4 molecule file, and Sequence pane with the primary sequence of the molecule. The three panes are integrated to reflect each other s actions. For a protein molecule, instead of Restriction Map folder as shown in Figure 2, Analysis folder is automatically created in the default setting in the Text pane. It includes molecular weight, isoelectric point and other physicochemical properties. With the Molecule Display window opened, you can load and edit the sequence and features, and perform various types of analysis such as polymerase chain reaction (PCR) primer design and molecule construction as described in the following sections. The Vector NTI module has some basic sets of tools such as creating a reverse complement molecule, finding open reading frames, translating a region or the whole DNA molecule, searching motifs and finding restriction enzyme sites. The Restriction Report function available in Vector NTI Advance (not in Vector NTI Suite) produces a list of non-cutting restriction enzymes. It does not have an option to generate other types of lists: for example, a list of single cutting restriction enzymes. If you see an asterisk appearing in a window title after the name of the molecule as in Figure 2, the molecule has been modified and needs to be saved in the database if the user wants to keep the changes. The asterisk was not observed in Vector NTI Suite for Macintosh, although the user manual described it so. At times Vector NTI seems to make some simple tasks unnecessarily tedious. For example, in order to create a reverse complement strand, you would think that just a simple click on a tool button would do the job. On the contrary, you need to open the Molecule menu, find the Operations submenu at the bottom, find again Advanced (DNA/RNA) submenu, and finally choose Make Reverse Copy to create a new file. Calculating the sizes of restriction fragments based on a particular set of restriction enzymes found by the Restriction Sites search function also requires multiple steps. Another shortcoming found is that it supplies no flexibility in saving the graphic file. The Camera function is used for this purpose. It provides options Picture Files (the PICT format for Macintosh) and Windows Meta Files (for Windows). Macintosh OSX users have another option for saving the file in PDF format using OSX s Print function. However, considering one reason to use Vector NTI would be for its graphic representation ability for molecular data, better compatibility with commonly used graphics software should be incorporated. Designing primers for PCR and sequencing Vector NTI can design primers for PCR, hybridisation probes and sequencing. Vector NTI takes into account almost all parameters that may affect the primer selection, which include parameters related to primer (eg melting temperature, T m, and percentage GC), amplicon, structure, pairs, similarity, 39 end, uniqueness, qualities and filters. It may not be easy for a novice to decide which values to set up with. In many cases, however, the default values seem to work just fine. A freeware Primer3, 7 for example, has been extremely useful in primer selection. However, Vector NTI offers some more attractive functions not found in other programs, which include designing primers for long PCR, alignment PCR and multiplex PCR. Moreover, PCR products can be saved to the database and used for molecule construction. Molecule construction The greatest strength of Vector NTI Advance and Vector NTI Suite lies in the plasmid construction. It is easy to create new molecules in Construction or Design modes. Construction takes the regular way of cloning; ie the user determines all the steps to be taken, which include defining the clone vector and inserts, choosing restriction enzymes, and selecting the methods of terminus modification. In the Design mode, new molecules can be created with the aid of & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER

5 Vector NTI built-in biological knowledge. The users need only define a list of donor and recipient fragments; the choice of restriction sites and terminus modification is left for Vector NTI to decide. The Design process generates a new molecule as well as a construction plan, which describes the best possible restriction sites and recombinant strategy. One important feature of the molecule design process is that the construction plan can be adjusted based on specific needs of the user by changing certain options. For example, the user may permit partial digests for constructing new molecules. The Fragment Wizard of Vector NTI provides a step-by-step guideline in defining a new molecule fragment, making the process of adding fragments quick and easy. The user can reconstruct a previously constructed molecule using the shortcut menu (right-click the molecule file) in the Database Explorer window. This Reconstruct feature is useful for constructing molecules that are similar to a previously constructed molecule and for reconstructing molecules that have failed a previous construction attempt. BioAnnotator (BioPlot for Macintosh) The BioAnnotator module enables the users to perform various basic DNA/ RNA and protein sequence analyses, displaying the results as linear graphics in the Graphics pane (Figure 3). BioAnnotator contains eight DNA/RNA analyses (eg GC content and melting temperature) and 50 protein analyses (eg antigenicity, hydrophobicity and polarity). Many of these analyses are related to each other. However, beyond providing a list of references in the manual, there is no description for each method, and users are left to speculate what those plots mean. For example, there are both GC content plot and nucleotide frequency distribution plot. How these plots are different (if they are) is not explained. AlignX and AlignX BLOCKS AlignX is the Vector NTI module that performs multiple sequence alignments and displays them with easily interpretable multi-colour graphics (Figure 4). Based on the popular Clustal W algorithm, 8 AlignX incorporates features including profile alignment, secondary structure Figure 3: BioAnnotator window 382 & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER 2004

6 Figure 4: AlignX window consideration, automatic consensus calculation, graphic display of a phylogenetic tree, dot matrix comparison and some alignment editing capabilities. The AlignX Display window is divided into four panes: Text pane for the description of each molecule included in the alignment project, Phylogenetic Tree pane for a phylogeny, Graphics pane to view plots of various types of analysis (eg alignment quality) and Alignment pane to display aligned sequences and the consensus sequence. The main features of AlignX are derived from a freeware Clustal X (the graphic and enhanced version of Clustal W). 9 Two improvements over Clustal X are the ability to display the neighbour-joining phylogeny graphically and the dot matrix for pairwise alignments. For both functions, separate software programs are required for Clustal X users. AlignX also allows the user to perform very basic manual alignment editing by shifting the position of gaps. What AlignX misses is Clustal X s flexibility to align only selected regions or molecules directly in the existing alignment. AlignX s profile alignment is limited and only one sequence or existing alignment can be used as the profile (a sequence or alignment for other sequences to be aligned against). AlignX supports only one alignment format, ie MSF format. In order for better compatibilities with other alignment and phylogeny programs, at a minimum, FASTA, Clustal W, PHYLIP and NEXUS formats need to be supported. AlignX BLOCKS is an independent program available in the AlignX module. It analyses and identifies localised sequence similarities (called blocks) among multiple protein sequences (Figure 5). It is an interesting tool. It will be useful for examining conserved regions, identifying functional domains and presentation of domain structures. It would be more useful if we were able to perform BLAST or Pfam search based on the block sequences identified. One small problem is in the use of words link and unlink in the Blocks menu (the AlignX menu for Vector NTI Suite). At first we thought simply that their functions are to link/ unlink domains along the sequence. They are used, however, for aligning domains (blocks) vertically across multiple sequences. Figure 5 shows a linked block alignment. & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER

7 Figure 5: AlignX BLOCKS window Sequence fragment assembly (ContigExpress) The ContigExpress module allows the user to assemble small fragments in text or chromatogram formats into longer contiguous sequences, ie contigs (Figure 6). The user needs to first create a ContigExpress project in order to work with ContigExpress. ContigExpress can recognise files in formats of GenBank, FASTA, ABI, SCF and some others. Surprisingly, it does not support the Applied Biosystem s ABD format. 10 The user needs to use, for example, the Staden package 11 to convert the ABD format to the SCF format before the file can be used with ContigExpress. Fragments with chromatograms can be edited directly and Figure 6: Contig Viewer window 384 & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER 2004

8 their changes can be tracked. Two methods, pairwise and linear, are used for assembly. The User s Guide explains that the pairwise assembly is best for assembling 10 or fewer fragments whereas the linear assembly is good for assembling 11 or more fragments. Similar to what was mentioned earlier, is that the algorithms used in these two assembly methods are not described. If the algorithms are available, it would help users understand their assembly results and compare them with other methods. The contigs can be saved in a GenBank, EMBL or FASTA format file. One important feature of ContigExpress is that it has many trimming functions for preassembly processing. These include vector contamination trimming, end trimming and Phred quality values trimming. GenomBench GenomBench is Vector NTI Advance s genome project application. There is no Macintosh version yet. In GenomBench, the user can search, retrieve and store data from several principal Distributed Annotation System (DAS) servers such as UCSC 12 and Ensembl. 13 GenomBench has a multi-pane interface including Overview pane, Info pane and Feature Map pane (Figure 7). GenomBench allows the user to search public databases, edit sequence and graphics, import and export annotated sequences, and send annotated sequences to Vector NTI. It also provides a means for mapping sequences onto genome regions using Sim4 14 or Spidey 15 genome alignment algorithms. GenomBench maintains its own local database for storing sequences and analysis results, which is independent from the Vector NTI database. GenomBench provides a seamless access for desktop computers to the DAS servers. Its application, however, is limited in that not many public DAS servers are available in default. The users need to create custom connections by themselves in GenomBench. Most genome-related DAS servers provide reference information only for model organisms. Certainly it would not provide much benefit to researchers who are working on non-model organisms. More functions (eg gene prediction) need to be integrated in order to serve for the general needs of genome projects. Figure 7: GenomBench window & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER

9 Internet tools Vector NTI takes a full advantage of internet. NCBI BLAST 16 similarity search is fully integrated into Vector NTI s graphic interface. It has its own dialogue window to manage various search options and BLAST Viewer to view the BLAST results graphically. One function that should be incorporated is the option to generate a multiple alignment quickly with the selected hit sequences. Several protein sequence analyses (eg NNPREDICT, TMpred), similarity search (eg PSI-BLAST) and pattern search (eg BLOCKS, PROSITE) can be accessed directly from the Vector NTI interface. The PFAM interface is available only on the Windows version. PubMed/Entrez Search is another Vector NTI s graphic interface program for the popular NCBI search tool. With Citation Table of Database Explorer and Citation View, Vector NTI provides a practical bibliography management tool complete with more than 50 journal reference styles, although the style selection has a strange preference (eg Nature, Cell and Proc. Natl Acad. Sci. USA styles are missing). Obviously all of these internet tools share the possible problems in networking connection and reorganisation of remote links. Frequent updates will be needed. 3D molecular viewer (3D-Mol) 3D-Mol is a stand-alone program included in Vector NTI. It can generate a graphic presentation of three-dimensional structures downloaded from the PDB (Protein Data Bank). 17 3D-Mol has many basic presentation capabilities with a simple clean interface. It has more flexibility than Cn3D 18 (freeware by NCBI). For example, 3D-Mol has a few useful functions as calculating distances and angles between atoms. Although it is not as powerful as Swiss-PDBViewer, 19 simply as a visualisation tool, 3D-Mol is sufficiently good and an easy-to-use software. On Macintosh, 3D-Mol requires the millions colour mode it did not run with the thousand colour mode. Only one graphic format (BMP both for Macintosh and Windows) is supported to save 3D images. Customising tools (Tools Manager) Vector NTI provides Tools Manager for customising items included in some menus. Tools Manager allows users to add new menu items to open internet links, start internet tools, or run programs and scripts. Because the number of internet links and tools included in Vector NTI is limited, this customisation capability is a welcome addition. It means Vector NTI could grow with users based on their specific research fields. CONCLUSION Computational tools and databases are essential for discovering knowledge embedded in the exponentially growing volume of biological data. Many sequence analysis suites such as GCG 20 are simply a collection of computer programs, each with individual input and output files, and it is not easy to manage different types of data and outputs. In this aspect, Vector NTI is unique and the Database Explorer provides a simple interface to manage and manipulate various types of data. The user can create new sub-bases to organise data used for different experiments and research projects. Vector NTI also has a good graphics presentation capability. Restriction maps and other figures can be quickly and nicely generated in Vector NTI. Vector NTI was also found to have several drawbacks. As mentioned earlier, it takes time for the first-time users to become familiar with the organisation structures. The use of its database system certainly makes the learning curve steep at the beginning; sometimes unnecessarily so. The Database Explore program integrates other modules through its menus. However, the opposite direction of integration is not supported. Once the users are in the AlignX module, for example, coming back to the database window is not easy, especially after 386 & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER 2004

10 opening many windows and modules. Adding a database icon on the tool bar of each module or adding a link to the database in each Window menu should solve this problem easily. Some of the programs such as dot matrix are only available in certain programs; some of the programs such as Alignment PCR are not very visible to the users. Some menus and submenus are confusing (eg editing restriction map via the menu View instead of Edit). In terms of display, some icons are too small (eg the icons +, in the Analysis List Setup window in BioAnnotator). Vector NTI can create a system administration nightmare quickly. After some hours of sequence analysis, users may, without knowing, end up running many different programs. All individual modules and related programs need to be closed one by one manually when users finish their work. This becomes a serious problem if the institution maintains a limited number of dynamic licences. A clean-up process should be incorporated when users log out or quit. Additionally, the manual should include the detailed list of available functions, so that users can find the necessary function easily. Instead of a single large manual, the tutorials and reference sections can be divided into two volumes. The reference part should include descriptions of each method instead of just giving a citation list. A comparison table of included functions between Windows and Macintosh versions should be provided on the web site. The number of supported output file formats needs to be increased especially for graphics formats. Finally, it would help first-time users if a template user database were created at the initialisation process in addition to the one containing all the examples. They would then not have to search their files from the large number of example files the installer copies during the installation process. Although it still has some problems and improvements are necessary, our overall impression is that Vector NTI is a wellbalanced integrated software package. Although the price is at the high end, at around US$1,500 for a single copy licence (plus US$500 for technical support), it comes with almost all basic computation tools routinely used in any molecular biology laboratory. It even includes a 3D viewer as well as a bibliography management program. Except for its nice graphics and bibliography capability, the majority of the functions have equivalent freeware available through internet (BLAST/PubMed server, Clustal X, Primer3, Swiss-PDBViewer, etc), and many more powerful programs are available through various web servers. However, for the laboratories and users who prefer to use a fully integrated environment without worrying about installing and using different software for each different task, and who are willing to learn and take the advantage of using an organised database system, this is a good package. With its customisable menus, the users can also incorporate their favourite tools if they are not supported in Vector NTI. Prospective users should take the advantage of the free trial licence, and spend some time to browse through the manual to see if the high cost and longer learning time are worthwhile for the functions it offers. Acknowledgments The authors were supported by the NSF EPSCoR program (EPS ). G. Lu is also supported by the NSF EPSCoR program (EPS ) and the INBRE Program of the National Center for Research Resources (P20 RR16469). Guoqing Lu, School of Biological Sciences and Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE 68588, USA glu3@unl.edu Etsuko N. Moriyama, School of Biological Sciences and Plant Science Initiative, University of Nebraska-Lincoln, Lincoln, NE 68588, USA emoriyama2@unl.edu & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER

11 References 1. Gilbert, D. (2000), Free software in molecular biology for Macintosh and MS Windows computers, in Misener, S. and Krawetz, S. A., Eds, Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totowa, NJ, pp Tippmann, H. F. (2004), Analysis for free: Comparing programs for sequence analysis, Brief. Bioinformatics, Vol. 5, pp Bioscience Technology (URL: 4. Chuba, P. J. (2000), Software review: Vector NTI Suite, J. Clinical Ligand Assay, Vol. 23, pp Wessner, D. R. (1999), Techview software. Planning plasmids, Science, Vol. 286, pp Invitrogen (URL: Rozen, S. and Skaletsky, H. J. (2000), Primer3 on the WWW for general users and for biologist programmers, in Misener, S. and Krawetz, S. A., Eds, Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totowa, NJ, pp (URL: 8. Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994), CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res., Vol. 22, pp Thompson, J. D., Gibson, T. J., Plewniak, F. et al. (1997), The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Res., Vol. 25, pp Applied Biosystem (URL: Staden, R., Beal, K. F. and Bonfield, J. K. (1998), The Staden Package, in Misener, S. and Krawetz, S. A., Eds, Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totowa, NJ, pp (URL: net/). 12. UCSC DAS Server (URL: genome.cse.ucsc.edu/cgi-bin/das/dsn). 13. Ensemble DAS Server (URL: servlet.sanger.ac.uk:8080/das/dsn/). 14. Florea, L., Hartzell, G., Zhang, Z. et al. (1998), A computer program for aligning a cdna sequence with a genomic DNA sequence, Genome Res., Vol. 8, pp Spidey (URL: spidey/). 16. NCBI BLAST (URL: Protein Data Bank (URL: Cn3D (URL: Structure/CN3D/cn3d.shtml). 19. Swiss-PDB Viewer (URL: us.expasy.org/spdbv/). 20. Saisanit, S. (2003), GCG sequence analysis, in Krawetz, S. A. and Womble, D. D., Eds, Introduction to Bioinformatics: A Theoretical and Practical Approach, Humana Press, Totowa, NJ, pp & HENRY STEWART PUBLICATIONS BRIEFINGS IN BIOINFORMATICS. VOL 5. NO DECEMBER 2004

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