Volume: 2DNASTAR Quarterly NewsletterIssue: 1
DNASTAR Quarterly Newsletter
Coming Events
4/8 Introduction of ArrayStar v2.16/8 Introduction of Lasergene v8.0
See Us At
4/17–4/19 Illumina User Group Meeting, San Diego, CA6/1–6/5 American Society of Microbiology, Boston, MA
4/23–4/24 Next Generation Sequencing, San Diego, CA6/12 Michigan State University, E. Lansing, MI
4/28–4/30 BioIT World Conference, Boston, MA6/25 University of Wisconsin, Madison, WI
5/31–6/3 European Society of Human Genetics, Barcelona, Spain 
Letter from the General ManagerTom SchweiVice President and General Manager

Dear DNASTAR Customers and Friends:

Many of you know DNASTAR as a company that has survived and thrived for 25 years producing software in support of sequence assembly and analysis. But that’s not all we have to offer. Last summer, we launched ArrayStar as our first product to support the microarray gene expression market needs. Within the next several days, we will be launching an updated version of ArrayStar, version 2.1, which includes many new features and functions.

Most importantly, ArrayStar 2.1 will include normalization methods for any data you want to bring into the product. Previously you could use the analytical and visualization capabilities of ArrayStar for normalized data. With ArrayStar2.1, you will also now be able to import, analyze and visualize raw data from various microarray platforms.

In addition, ArrayStar 2.1 will include a new import wizard to give you more choices when you are bringing data into the program. We have also substantially enhanced the searching capability within the feature descriptions that can be loaded into ArrayStar.

Finally, we’ve improved the file and memory handling capabilities of the product to allow for larger data files to be imported and processed within ArrayStar. Overall, in the nine months since we first launched ArrayStar, we have been encouraged by the response of the market to this new product and we feel good about the improvements to the product that we are able to make in this new release. We encourage you take our free trial version of ArrayStar for a test drive to see if it meets your needs.

We continue to work hard on other product releases as well, including new offerings in the area of sequence assembly and analysis, including substantial new offerings supporting next generation DNA sequence capabilities. We are about to embark on a beta test for Lasergene 8.0, which will include many substantial new features supporting next-gen sequence assembly and analysis, the incorporation of primer design directly in virtual cloning projects, improved coordination with our GenVision genome visualizer product and many other enhancements. If you are interested in participating in our Lasergene 8.0 beta test, please contact me or send us a note at support@dnastar.com.

All of us at DNASTAR continue to be honored and feel privileged to serve the life science community in the important work you are doing. As always, please let me know if we can improve the way in which we are serving you.

Sincerely,

Thomas E. Schwei
Vice President and General Manager

NewsDNASTAR Expands International Distribution Into China

The growing importance of the Peoples Republic China and Hong Kong in genomics research is evident. To better serve customers and expand the company’s presence in these markets, DNASTAR signed a distribution agreement with BGI LifeTech Ltd in Beijing.

BGI LifeTech (http://www.bgilt.com) is a sequencing service and product distribution company that has been in business for 6 years. Its headquarters is located in Beijing, however, it has sales representatives covering major cities and research centers throughout China and Hong Kong. The company is a spin-off from the Beijing Genomics Institute.

With the growing interest in advanced sequencing and gene expression data analysis, the company believes that the expansion of its distribution network with the inclusion of BGI LifeTech will significantly strengthen its position in this market.

Marketing Director Bob Steinhauser stated “it is our belief that having a local representative that is knowledgeable in sequencing and gene expression analysis will significantly improve our business coming out of China. BGI LifeTech is well connected in the mainland genomics business and we believe there is a good fit between our 2 companies. We are very excited about the potential for our software with a solid company like BGI LifeTech.”

Training of the personnel at BGI LifeTech is underway at the present time and it is expected that they will be up-to-speed with the different products shortly.

ApplicationsSNP Reporting

A key component to the SeqMan Pro assembly module in Lasergene provides users the option of SNP discovery and analysis. Central to this is the SNP reporting function that provides in table format information on each SNP identified in the analysis. From the All Found and Summary reports, users are able to review each SNP identified. The explanations following explain how best to access and navigate through the SeqMan SNP reports.

I. Using the ALL FOUND SNPs Report

Click to enlarge

A row in the All Found SNPs report lists information for a single SNP base in a sample sequence.

To view the report when the SNP Report window is already open, click on the All Found SNPs tab.

To view the report when the SNP Report window is not already open:

  1. Choose a contig by one of the following methods,
    • In the Summary View, select a contig name in the top pane.
    • Display the Alignment View for the contig.
    • Display the Strategy View for the contig.
  2. Select SNP»SNP Report. The SNP Report window displays the All Found SNPs report.

Navigating through SNPs from the All Found SNPs report

To navigate through the SNPs in the Alignment View:

  1. Click on a row in the All Found SNPs report. The SNP base corresponding to the row is selected in the Alignment View.
  2. Click on the up or down arrow key on the keyboard to move to the previous or next SNP in the report.

Switching from the All Found SNPs Report to the Alignment View

To switch to the Alignment View, double click on a row in the All Found SNPs report. The Alignment View is displayed with the SNP base corresponding to the row selected.

Sorting columns in the All Found SNPs Report

Click on a column header to sort the rows by that column. Click on the triangle on the right of the column headers to switch between ascending and descending sort orders.

Click to enlarge

II. Using the SNP Summary Report

A row in the SNPs Summary report summarizes information for all of the SNP bases in an aligned column.

To view the report when the SNP Report window is already open, click on the SNPs Summary tab.

To view the report when the SNP Report window is not already open:

  1. Choose a contig by one of the following methods,
    • In the Summary View, select a contig in the top pane.
    • Display the Alignment View for the contig.
    • Display the Strategy View for the contig.
  2. Select SNP»SNP Report.
  3. Click on the SNPs Summary tab.

Navigating through SNPs from the SNP Summary Report

To navigate through the SNPs in the Alignment View:

  1. Click on a row in the SNP Summary report. The SNP column corresponding to the row is selected in the Alignment View.
  2. Click on the up or down arrow key on the keyboard to move to the previous or next column containing SNPs in the report.

Switching from the SNP Summary Report to the Alignment View

To switch to the Alignment View, double click on a row in the SNPs Summary report. The Alignment View is displayed with the column corresponding to the row selected.

Sorting columns in the SNP Summary Report

Click on a column header to sort the rows by that column. Click on the triangle on the right of the column headers to switch between ascending and descending sort orders.

ArrayStar 2.1New Features Further Simplify Microarray Gene Expression Analysis

Scatter Plot — Click to enlarge

Ten months ago DNASTAR introduced ArrayStar v2.0 software for microarray gene expression analysis applications. Keeping with the company’s philosophy of introducing frequent upgrades to its main software products, ArrayStar v2.1 will be introduced in mid-April.

A number of improvements have been added to this version to further simplify gene expression tasks. Improvements have come in several different areas focusing on

  1. expanding the number of microarray gene expression platforms where the software is compatible
  2. expanding the size of data sets that can be analyzed
  3. including Normalization methods that can be easily performed
  4. providing MAS5 chip summary statistics for Affymetix data
  5. providing support for gene classification/ontology applications
  6. permitting easy filtering by gene classification

Data Preprocessing — Click to enlarge

Files can be imported into ArrayStar in a variety of formats. It fully supports the import of Affymetrix *.chp files and also can download and import annotation files for the corresponding array directly from Affymetrix. Nimblegen *.call or *.calls files can also be imported. Illumina BeadStudio export files and any arbitrary delimited text file containing microarray data can be imported into ArrayStar using the Data Import Wizard.

ArrayStar v2.1 can be used with either normalized or raw data requiring normalization. The software provides an easy-to-use Import Wizard that requires the user to only select the normalization method from a drop down menu and then to enter any selected modifications they want for their analysis as the image below illustrates.

ArrayStar v2.1 software enables researchers to perform a wide range of analyses on their data. Multi-functional scatter plots can be generated that allow the user to select groups of genes for analysis. Much information can be extracted from the scatter plot. The graph to the left illustrates a group of selected genes (white) that have been selected from the overall experiment. Any combination of genes can be clustered for additional analysis. Passing the cursor over any of the genes automatically provides gene ontology and pathway information. Prompts in the lower right hand corner provide From the gene set a wide range of information can be can be quickly obtained. For example, Gene Ontology and pathway information can be easily obtained for the selected set.

Heat Map — Click to enlarge

Heat maps can be generated on any gene set designated. Information on specific genes and on their relationship within the cluster is shown. A gene tree on the left of the Heat Map shows the clustering of genes as calculated by the selected clustering method. Clicking on a node in the tree allows the user to quickly select that sub-tree of genes.Names of different genes are displayed on the right side of the Heat Map.

Advanced Filtering — Click to enlarge

The Info Pane on the right of the Heat Map gives information about the data displayed. A histogram of the data distribution in the Heat map is above the color scale. As selections are made on the Heat Map, the selected genes are shown in gray within the histogram. Minimum and maximum expression level values are also shown beneath the color scale.

Of particular help to users is the Actions section in the lower right corner of the screen. This provides an easy prompt to users regarding possible follow-up actions they may take while also providing them with a quick method to perform them.

Another new feature coming with ArrayStar v2.1 is the ability to filter through the Advanced Filter to identify genes of interest based on gene classification/ontology values among others.

Gene Table — Click to enlarge

The Gene Table view (shown below) contains detailed information for every gene in your project, including both the expression data (e.g. signal intensities and fold change values) as well as any annotations that are available from imported sources, such as the gene name(s) and gene ontology. Some annotations have special features, allowing you to hover over a term for more information, or click on a hot link to view more detailed information online. Any gene subsets being investigated are indicated in the Gene Table, allowing you convenient access to key tabular information for the genes being visualized by other tools in the package. The data in the Gene Table may be searched, printed, copied as a text file, or exported as a tab-delimited or comma-delimited text file.

ArrayStar v2.1 continues to provide users with many statistical tools and visualizations to help further the analysis of your experiments. For example, users will be able to easily generate Line Graphs that illustrate relative gene expression levels within the gene set. This can be very helpful in identifying regulation patterns and relationships. Statistical methods allow users to quickly examine relevance of actions performed. And much, much more!

For additional information, check the DNASTAR website after April 15th for additional technical information and details.

Tips and TricksAnne StoverTechnical Support Specialist

  • Simplified Steps for Large Assembly Projects in General
  • Recommendations Specific to Roche 454® Data Sets
  • Recommendations for Assembling Sanger and 454® Datasets Together

If you have particular Tricks for using Lasergene that you believe would benefit your colleagues, let us know so we can share them.

Simplified Steps for Large Assembly Projects in General

Click to enlarge

SeqMan Pro is the assembly tool of choice for numerous types of projects on various desktop computers. Since data types and computers vary, your experience using SeqMan Pro will depend on several variables: the data used (the number of reads, read length, and depth), parameters chosen, and your computer’s RAM. If you are not able to complete your assembly with our suggestions below, please contact DNASTAR for additional help and direction. Larger, more complicated Assemblies using Roche 454, Illumina and/or Sanger platforms may require the use of SeqMan Genome Assembler that has been designed specifically to handle issues related to Next Generation sequence assemblies.

SeqMan Pro offers a wide variety of both Pre-Assembly and Assembling parameters that can be adjusted, depending on the type of data set you are trying to assemble. Parameter settings can be accessed by selecting Project»Parameters from the SeqMan Pro menu bar. Adjusting parameter settings can greatly influence both the accuracy and speed of your assembly.

General Recommendations for Assembling Large Data Sets

  • Use the Pro Assembler method instead of the Classic Assembler by going to Project»Parameters»Assembling and choosing Use Pro Assembler.
  • Make sure that the Don’t add single sequence contigs option is selected in the Pre-Assembly parameters. Allowing single sequence contigs to be added to your project can greatly decrease the speed of the assembly. Access this dialog by clicking on the Options button from the Unassembled Sequences Window. This option is on by default.
  • Always do vector scanning and quality trimming. The default Medium Quality Stringency trimming works well for most datasets.
  • Increase the consistency of sequence depth throughout your assembly by the type of coverage you expect:
    • If you expect fairly even coverage, select the Use Repeat Handling option under Assembling parameters. Choose the Fragment Length radio button and enter the length of the genome or fragment being assembled.
    • If you expect deep coverage, select the Use Repeat Handling option under the Assembling parameters. Choose the Fixed radio button and enter your expected coverage size.
  • When adding sequences to an existing assembly (iterative assembly) and adding only a sparse number of sequences, it is usually better to turn off the “Use Repeat Handling” option.
  • Changing the Match Size assembly parameter is not advised as it will increase false joins.
  • Upon completion of a de novo assembly, all of the single sequences that did not enter a contig remain in the Unassembled Sequences window and can be viewed by selecting Sequence»Add. To add these sequences to the existing assembly, first adjust the parameters (by selecting Project»Parameters), and then click Assemble from the Unassembled Sequences window.

REMEMBER: Increasing your computer’s memory and processor speed will influence the speed of your assembly. The more RAM and the faster the processor, the faster your assemblies will complete. On Windows, increasing your virtual memory may help.

Recommendations Specific to Sanger Data Sets

  • Select Use Pro Assembler under Assembling parameters (Project»Parameters) and use the default values, as shown on the picture above.
  • Enter in trace files for viewing chromatograms.
  • Always do vector scanning and quality trimming. The default Medium Quality Stringency trimming works well for most datasets.

Recommendations Specific to Roche 454® Data Sets

  • Enter the .sff file. The .sff format includes the base calls, qual scores and the flowgram data. In the Unassembled Sequences Window, the .sff file will be listed as a Flow type.
  • Set the Minimum Sequence Length assembling parameter to a length reasonable for your data set. The default of 100 is often too long for 454® data. A value of 70 is typically a good place to start.
  • Set the Maximum Mismatch End Bases assembling parameter to 0. This represents the number of bases from an end where mismatches are not counted when calculating pairwise similarity.
  • Set the Match Spacing assembling parameter to 15. The default of 150 is often too large for 454® data.
  • Set the Maximum Expected Coverage parameter, found under Project»Parameters»Strategy Viewing to the maximum depth that you expect in your assembly. After assembly, areas exceeding this maximum value will be indicated in the Strategy View by thick, red areas in the Coverage bar.
  • SeqMan Pro does not handle 454® dual end datasets.
  • Reading long homopolymeric regions accurately is a limit of 454® technology. Please consult the 454® website for additional information.

Recommendations for Assembling Sanger and 454® Datasets Together

  • Enter both Sanger and 454® data at the same time.
  • It is not recommended to assemble contigs with contigs.
  • Starting with a high stringency setting for the Minimum Match Percentage (85) and then adding the sequences that did not go in using a decreased percentage (80) can be effective.
  • If you have a complete Sanger assembly that you are confident with and you are using 454® reads to fill in areas of light coverage, or vice versa, you can use an iterative approach starting with the more complete data set. If you use the iterative approach, turn off Repeat Handling for the second run.
  • It is extremely important not to forget to perform vector trimming on the Sanger sequences!

Featured ModulePrimerSelect — Helping in the Design of Your Primers
Primer Workbench — Click to enlarge

PrimerSelect is designed to help users design primers and probes for PCR, sequencing and hybridization experiments. Enter your own primers or let PrimerSelect generate a sorted list of suggested primers for all or part of your DNA, RNA or back-translated protein template. You can accept PrimerSelect's top choice, evaluated on the thermodynamic properties for annealing reactions, or view all of the options and choose alternatives based on your specialized knowledge.

PrimerSelect includes a WorkBench (see below) where you preview the effects your choices will have on reading frames and restriction sites. Customizations possible with the module to primer design include:

  • The ability to adjust parameters such as primer and product length, primer locations, Tm, hairpins or use default settings
  • Edit template sequence and share newly created features through integration with SeqBuilder and other Lasergene modules
  • Determine the best primer or probe for differing conditions
  • Analyze existing primers
  • Create your own primers
  • View the effects on restriction sites and translations when editing primer sequences
  • Select from a list of alternative codons for silent mutations or specific residue changes
Located Primer Pairs — Click to enlarge
Summary Report
 — Click to enlarge

The Located Primer Pairs window (shown below) shows a list of primer pair candidates. The best candidate is listed at the top of the table. The example shown also shows Score, dTM, Ta–Tm and primer length information. Placing the cursor on the small figure above the word length and dragging left or right provides additional advice on each of the primer pairs.

Below is an example of a Composition Summary Report that can be generated from the Report section in the menu. Similar reports can be generated on Primer Hairpins, self-dimers and pair dimers.

PrimerSelect permits users to create a new primer and to save it into a catalog for future reference. Favorite primers can be placed in special lists to simplify their location at later dates. Oligonucleotide request forms can be created with the active primers entered automatically to further save time and minimize error possibilities. Through its NetSearch menu, PrimerSelect permits searches for similar sequences in BLAST databases. If you already know the locus name or accession number of a sequence in NCBI’s Entrez database, you may open it directly in PrimerSelect. For additional information regarding PrimerSelect, either check our website under Lasergene or contact DNASTAR Technical Support at info@dnastar.com.

Customer ProgramDNASTAR Lasergene Configurations Included on GSA Purchasing Agreement

DNASTAR announced that it has concluded a contract with the General Services Administration of the Federal Government that will permit federal government buyers to purchase selected configurations of its Lasergene software through a contract negotiated with GSA. The company believes that it is first organization to offer DNA and protein sequence analysis software through such an arrangement with the Federal Government.

Under terms of the agreement, qualified purchasers will be able to purchase selected configurations of Lasergene through terms that will permit each such buyer to take advantage of the overall purchasing power of the Federal Government. In addition to preferable purchasing terms, the purchasing process will be significantly simplified for all buyers using the program.

“US Government related purchases of our software are important parts of our growing business”, stated Marketing Director, Bob Steinhauser. “Anything we can do to assist our government customers in the purchasing process is an important interest for us. We look forward to going forward with the agreement and being able to work more closely with the vast number of Agencies and buying groups that can utilize such an Agreement to simplify their buying process.”

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