DNASTAR Newsletter, Volume 1, Issue 4

Dear DNASTAR® Customers and Friends:

As we begin 2008, it is time to both look back at past accomplishments and look ahead to future prospects. At DNASTAR, we are able to do both by discussing one key technology area — next generation DNA sequencing and software to support data generated by the new instruments that have taken hold in the market during 2007.

2007 was a good year for DNASTAR related to next-gen technologies. We were able to make a significant impact on the market for next-gen software throughout 2007 by making available SeqMan Pro, which can handle Sanger and 454® data files, including visualizations of both trace files and 454 flow-grams. With this module of Lasergene, we have served many customers working with bacterial genome assembly, SNP detection or other similar applications of next-gen technologies on their Windows or Macintosh desktop computers. In fact, in 2007, we realized our first SeqMan-only site license from an institute that needed the next-gen capabilities of SeqMan Pro and decided to make it available to all of their laboratory scientists.

Also, we are now making available an Early Release version of SeqMan Genome Assembler, our next-gen tool for handling Illumina/Solexa®, 454® and Sanger data. Due to numerous requests from the scientific community, we took a step that we’ve never before taken of making this tool available in an Early Release version. The algorithms have been tested and we have a fully documented scripting language for running the program. We are making it available to scientists before putting all the finishing touches on the program in the interest of making this important tool available to those who need it. If you have next-gen data and don’t know how to process it for your intended application, give us a call. We should be able to help you.

As to the future, all we can say is that it has never been brighter for scientists working with DNA sequences. The possibilities being opened up by the next-gen technologies are phenomenal. New approaches to solving old problems are coming to the forefront daily. At DNASTAR, we have a strong commitment to continuing to evolve our products and programs to support these new applications of DNA sequence technology, while also supporting the tried and true applications.

I hope you had a successful year in 2007 and, on behalf of all of us at DNASTAR, I want to wish you all the best in 2008. Happy New Year!

As always, please let me or anyone else at DNASTAR know if we can improve the way in which we are serving you.

Sincerely,

Thomas E. Schwei
Vice President and General Manager

SeqMan Pro gives you the ability to screen for contaminant sequences in your assembly. For example, if you are sequencing yeast DNA inserts that were cloned in E. coli, you may want to remove any contaminating E. coli DNA from the project prior to assembly. To do this, follow the steps below:

1. Store sequence files of known contaminants in the Contaminant Seqs folder found in the following location:

Windows Vista^TM: C:\Users\Public\Public Documents\DNASTAR\Lasergene 7 Data

Windows® XP/2000:

C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 7 Data

Macintosh®: Hard Drive:Applications:DNASTAR:Lasergene

2. Within SeqMan Pro, select Project»Contaminant Sequences. Files stored in the Contaminant Seqs folder appear in the Available window on the left:

3. Specify known contaminants by selecting the appropriate sequences from the list on the left and clicking Add.

Selected contaminant sequences move to the Scanned list on the right.
If you decide not to use one of these contaminants, highlight its name in the right window and click Remove.

4. Click OK to save changes.

5. If desired, you can edit the Contaminant Screening parameters by selecting Project»Parameters» Contaminant Screening:

6. Type in the Minimum Number of Matches required to mark a given sequence as a contaminant. match in this instance is any 16-mer in common. Then, click OK.

7. To screen for contaminants, add sequences to the Unassembled Sequences window:

8. Click Options in the top right corner of the window, and check Remove Contaminant Sequences:

Click Scan Selections to search for contaminants against highlighted sequences only.
Click Scan All to search all sequences.
Click Scan Later to postpone contaminant removal until clicking Assembly.

Tip: Unlike vector trimming, which eliminates only the segments of reads that match vector sequence, contaminant screening prevents the whole read from entering the project. Therefore, you should use this feature cautiously. If your project is large, you may want to increase the minimum number of matches to ensure that chance similarities between contaminant and target sequences do not eliminate informative data from your assembly.

Click to enlarge
Next Generation sequencing technology platforms are providing researchers with tools necessary for a wide range of potential applications including genome re-sequencing, deep sequencing and de novo sequencing, to name a few. Because of the large volumes of data generated with each run, economical software to assemble and analyze this data is important to the researcher. DNASTAR has been approached by many users of different Next Generation sequencing technologies and urged to introduce an assembly tool that can assist users with their data handling needs.

The Early Release version of DNASTAR’s new SeqMan Genome Assembler provides its users with a tool for handling data generated from 454®, Illumina® and Sanger methodologies for large bacterial genome size projects. It works in conjunction with the SeqMan Pro and SeqBuilder modules in Lasergene to utilize a broad range of Lasergene’s display and analysis capabilities.

SeqMan Genome Assembler is a command-line application that uses a unique algorithm to assemble fragment data sequenced using Illumina®, 454®, and Sanger technologies. It offers complete flexibility in adjusting assembling parameters to meet the needs of your specific data set. The default parameter settings are included to assist in getting started especially with most Illumina® assemblies, while the Optimizing Assembly Parameters features allow users to adjust parameters specific to the different types of data sets they might have.

As the user, you may also decide on a number of preprocessing options, including vector and end-trimming, and the ability to exclude known repeats and contaminant sequences, such as primer reads, from your assembly. Illumina® data most commonly requires a template sequence (also called a reference sequence) for assembling in SeqMan Genome Assembler. Multiple templates can also be used if desired. 454® and Sanger data sets can be assembled de novo, or with a template sequence.

Annotating template sequences in SeqBuilder for known SNPs, CDSs, and other features prior to assembly can be performed to further enhance the analysis of identified putative SNPs in SeqMan Pro. Once sequences have been assembled and saved, results may be viewed in SeqMan Pro.

During the setup of SeqMan Genome Assembler, the user has the option of setting maximum depth of coverage value. If set, the user may visualize the areas where the coverage depth is exceeded by going to the Strategy View in SeqMan Pro and looking for the thick red areas.

SeqMan Genome Assembler offers three output options for saving your finished assembly: the SeqMan Proproject file format, as a Phrap assembly, and in FASTA format. Following assembly in SeqMan Genome Assembler, saved projects can be viewed in SeqMan Pro for analysis, including identifying SNPs and analyzing coverage.

SeqMan Genome Assembler can also export a report summarizing your assembly statistics, including the number of assembled/unassembled (matched/unmatched) sequences and contigs in your project, the parameters used, the average quality scores, and the number of sequences excluded from the assembly due to exceeding the maximum coverage parameter.

Although the upper limit for project size depends on many factors, including the amount of your computer’s RAM and processor speed, a computer meeting our recommended requirements should be able to use SeqMan Genome Assembler to easily assemble one lane of Illumina® data (about 4 million reads), one million 454®reads, or 200,000 Sanger reads.

For more information on SeqMan Genome Assembler either contact your DNASTAR sales representative or distributor or go to our website at www.dnastar.com.

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

Using SeqBuilder Images for Publication
Click to enlarge

SeqBuilder offers high-quality linear and circular map images that you may want to export into another application, such as Microsoft® Word, PowerPoint, or Adobe® Illustrator®.

SeqBuilder’s Copy as Picture command allows you to do this easily.

To use this feature:

1. Open the graphical view you want to copy

2. Choose Edit»Select All and then Edit»Copy as Picture. The graphical view is then copied to your clipboard and can be pasted into another application.

NOTE: In Microsoft® Word and PowerPoint, be sure to select Edit»Paste Special and then select Picture (Enhanced Metafile).

Realigning Residues in MegAlign

In MegAlign, after completing a multiple alignment, you may manually realign portions of your alignment using the Straighten Columns, Shuffle Right and Shuffle Left palette tools, found on the left side of your worktable:

Straighten Columns		Aligns selected residues from different sequences by inserting gaps to the left of the selected residues.
Shuffle Right		Moves residues from the left end of the gap to the right end.
Shuffle Left		Moves residues from the right end of the gap to the left end.

To manually realign residues:

1. Make the Worktable the active window.

2. Scroll to the region that you would like to realign.

3. Straighten columns by selecting the residues you want to align, then clicking the Straighten Columns tool.

4. Shuffle residues by selecting regions in which residues and gaps are interspersed. To slide residues to the right of the highlighted regions, click the Shuffle Right tool. Use the Shuffle Left tool to slide them in the opposite direction.

5. Manually edit the alignment by adding gaps using the space bar, or removing gaps using the Backspace key. Note that Backspace can only be used to remove gaps, not residues.

6. If necessary, you may undo realignment by using Edit»Undo.

Tip for Effective Entrez Searches
Entrez Query — Click to enlarge

All Lasergene applications allow you to search for sequences by querying NCBI’s Entrez Database. This option can be accessed in any Lasergene application by selecting Net Search»New Text Search.

The Entrez Query interface allows you to create a Boolean query, which you can use to search multiple fields for multiple search criteria.

If you receive fewer results than you expect from your search, try putting parentheses around your search criteria.

For example, if you are searching for protein sequences containing the term Fasciclin I, try searching for it as (Fasciclin I). Doing this should yield the maximum number of results.

Click the following link to NCBI’s website for more information on searching with parentheses.

Each issue of the DNASTAR newsletter features a short article written by a research scientist in the field of molecular biology. The intent of the article is to provide readers with additional information from their colleagues about work they are doing using sequencing as a key element. For inquiries and additional information on the article, email DNASTAR.

The removal of two dams on the Elwha River in Washington State and the restoration of the river and salmon runs, represents the largest dam removal and most complete river restoration ever conducted. Moreover, this project is ranked as the second most urgent restoration priority by the National Park System.

The dam removals provide an excellent opportunity to establish a unique opportunity for undergraduate students to play a significant scientific role in a major ecological/natural resource management issue. Undergraduate students at Peninsula College (PC) in Port Angeles, Washington are involved in an NSF funded research project (NSF-DBI-0452328) to study the Elwha ecosystems throughout the year, characterizing the current status and within-year variations of chemical, physical, nutrient, biological, and ecological conditions in the watershed, and then comparing these to conditions post-dam removal.

Dr. Bill Eaton (Principal Investigator on the NSF grant) has been working with students to examine variations in the rDNA gene diversity and microbial community structure within the biofilm on the rocks in the Elwha River below, between, and above the dams, and from the Quinault River. He and his students are using PCR methods, universal rRNA primers, and standard cloning methods to generate rDNA sequences within clone libraries from the three reaches of the Elwha River and the Quinault River. The sequences are then being examined using the Lasergene SeqMan Pro software program to determine the different Operational Taxonomic Units (OTU) within the clone libraries. These sequences are then compared to known submitted sequences within the NCBI database to determine taxonomic status of the rDNA sequences. Relationships between the OTUs and the microbial community structure have been studied with the help of the Clustal W and Bootstrapping capabilities within the MegAlign program.

The goal has been to find potential habitat variation indicators (PHVI) within the clone libraries derived from samples collected from the different areas. The preliminary results so far have been interesting. Initially, the 16s rDNA clone sequence analysis of the three clone libraries yielded 27 unique clone types, each representing a unique taxonomic group as determined by having more than 95% DNA sequence identity with sequences previously submitted to the NCBI database. These were composited into 12 major phylogenetic groupings: alpha, beta, delta, epsilon, and gamma Proteobacteria, Bacteroidetes, Cyanobacteria, Verrumicrobia, Deinococcaceae, Micrococcaceae, Planctomycetaceae and Bacillaceae.

Dr. Eaton and his students then examined the abundance and distribution of these bacteria within the different reaches of the river, looking for PHVIs. Their preliminary results are encouraging and summarized as follows.

Preliminary Results:

There was significant variation in the amount and percent of beta Proteobacteria in the samples collected from below the dam: 62.5% (25/40) clones from below the dams were from the beta Proteobacteria group. Only 41.9–44.2% (18/40-19/45) of the clones from the other two reaches of the Elwha River and the Quinault River were within the beta Proteobacteria group.
There was significant variation in the amount and percent of Bacteroidetes in the EU samples collected from above the two dams: 34.9% (15/45) clones from above the dams were from the Bacteroidetes group, while this group was represented by only 5% (2/40) and 16.3% (7/43) of the clones from the other two reaches and only 11.6% (5/40 of the clones form the Quinault River.
Epsilon Proteobacteria clones were only found in the clones form the Quinault River (20.9%; 9/40) and none from any of the Elwha River clones.

While the results are interesting, additional work is on going to analyze more clones from the different sample’s sites to develop more clear PHVIs that may provide a new monitoring tool once the dams are removed. Perhaps more importantly, this study, and the use of the Lasergene software package has helped undergraduate students develop a better understanding of molecular microbial ecology and the “mysteries” of DNA sequence analysis.

SeqBuilder is the Lasergene module that enables users to edit nucleic acid and amino acid sequences and view the sequences and related items in a variety of ways. SeqBuilder's window can be split into multiple panes, permitting more than one view to be displayed at any time. The possible views include:

a residue-specific view
a linear map
a circular map
a compact map of restriction cut sites (mini map)
as well as a list of features
a summary of all restriction cut sites, and any comments

Each view lists a number of items that can be individually turned on or off including features, restriction sites, translations in all six reading frames, ORFs, and rulers. Many of these items can also be positioned and modified to emphasize certain characteristics. Dynamic links between the sequence and its annotations automatically update feature coordinates when you edit a sequence. Available functionality also includes the ability to reverse complement, translate, back-translate sequences, identify ORFs and perform BLAST and Entrez text searches directly to NCBI.

The power and versatility of SeqBuilder lies in its editing capabilities that are also integrated into many other Lasergene modules. Edits made in SeqBuilder will be shared automatically with other Lasergene applications that are analyzing the same data, making SeqBuilder an integral member of the powerful Lasergenesuite.

SeqBuilder is an especially powerful module when used in conjunction with DNASTAR’s new SeqMan Genome Assembler when you are working with large assembly projects using either 454® or Illumina® Next Generation sequencing platforms or Sanger methods. Prior to the assembly in SeqMan Genome Assembler, it is possible to annotate your template sequence in SeqBuilder for a number of features including:

known SNPs/variations
exon
CDS
miscellaneous RNA
Rep origin
rRNA
tRNA

As shown in the image on the right, virtual cloning projects can easily be created and edited in SeqBuilder. Vector catalogues can be created and modified. Dynamic links between the sequence and the annotation provides automatic updates to feature coordinates as a sequence is edited. In addition to restriction cloning, SeqBuilder also supports TA Cloning, Gateway® Cloning and Directional TOPO® Cloning.

ORF Viewing and Analysis — the image on the left shows the ease which SeqBuilder can be used to identify ORFs and to analyze changes made instantly with any sequence change.

DNA can be translated or proteins can be easily back translated using either provided genetic codes or ones the user has generated independently.

SeqBuilder is a key module in Lasergene. Its flexibility make it one of the most useful modules of Lasergene for researchers. To learn more about SeqBuilder and its capabilities and the other modules of Lasergene v7.2, go to www.dnastar.com/products/lasergene.php.

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