DNASTAR

What’s the difference between SeqMan NGen, SeqMan Ultra, and SeqMan Pro?

SeqMan NGen and SeqMan Ultra are two applications that work together to assemble and analyze both Sanger and NGS sequencing data. SeqMan NGen is the application that assembles and aligns the data. SeqMan Ultra is one of the applications that allows you to analyze and edit the assembly produced by SeqMan NGen. (Additional applications within Lasergene Genomics are available for analyzing NGS projects and more complex Sanger assemblies).

SeqMan Ultra is the new face of SeqMan Pro, our longstanding sequence assembly application, complete with a new modern, intuitive interface, as well as lots of performance improvements under the hood. SeqMan Ultra is a 64-bit application, which means when compared to the 32-bit SeqMan Pro application, it offers faster project opening, better performance for analyzing large files, increased capacity, and compatibility with macOS 10.15.  SeqMan Pro will still be available in Lasergene 17, but will eventually be completely replaced by SeqMan Ultra.

How is the consensus sequence calculated?

SeqMan NGen uses a proprietary “Trace Evidence” algorithm for Sanger sequence assemblies that has significantly better consensus calling accuracy than the “Majority” method used by most consensus calling programs.

How do I get best possible results in my de novo Sanger assembly?

De novo assembly is sensitive to low quality or untrimmed vector sequences. Our sequence assembly tool provides fast and accurate trimming options based on quality and vector (or other contaminant) sequences.

Can I assemble multiple samples for variant analysis?

Yes. When you set up your Sanger sequence assembly project in SeqMan NGen, choose “Multi-Sample” when adding your input sequences. You can then groups your reads into experiment groups, making it easy to compare samples in the finished project.

Can I edit a de novo Sanger sequence assembly?

Yes. A de novo assembly run in SeqMan NGen produces a fully editable project file (.sqd) that can be opened in SeqMan Ultra, where base-level and contig-level editing can be carried out.

What file types can I import to assemble?

For Sanger sequence assembly projects, SeqMan NGen accepts output from most Lasergene applications as well as 454, ABI, ALX, FASTA, GCG, GenBank, Phred, SCF, Sequencher, .eff, .fof, .txt and .zip files.

When should I assemble sequences using a reference?

A known sequence can be used as a backbone/template/reference sequence upon which to build your sequence assembly. This lets you perform the assembly with lower average sequence coverage, thus saving a substantial amount of sequencing effort. Simply choose a reference-guided workflow when you set up your project, and you will be prompted to input a reference sequence.  If no reference sequence is available for your organism, try using one from a closely-related organism.

Can I add new data to my sequence assembly?

Yes, you can add new sequences to a draft genome assembly in SeqMan Ultra for the purpose of scaffold gap closure. Simply select two adjacent contigs in a scaffold and then choose Contig>Add sequences to close gap to add one or more sequences to close the gap.

How do I organize contigs in my sequence assembly?

There are several options for organizing contigs in an assembly. If your input data is paired, you can use Contig>Order Contigs to automatically create scaffolds based on the paired data. You can also select contigs and use Contig>New Scaffold to manually create scaffolds or simply drag and drop contigs to reposition in the Explorer panel.  If you use the Hybrid reference-guided/de novo assembly workflow, contigs will automatically be placed in scaffolds based on the reference positions.

How do I align/join contigs in my Sanger sequence assembly?

Select two or more contigs in the Explorer panel and use Contig>Align contigs end to end to align a multiple contigs to each other. If your contigs are in a scaffold, this alignment will preserve the position within the scaffold and greatly reduce the chance for misaligning contigs at repetitive elements. You can also manually align contigs using Contig>Force Join if you know that two contigs should be merged, but there is insufficient overlap for successful alignment.

What options do I have for editing contigs and contig consensi in my sequence assembly?

SeqMan Ultra offers several options for large scale contig editing, including: splitting (where mismatches occur), reverse complementing, deleting, and micro-editing  to remove errors from the consensus. The navigation toolbar allows you to quickly and efficiently search the consensus for conflicts to manually edit.

How can I export my completed sequence assembly?

From SeqMan Ultra, select Contig > Export Consensus and choose from the following options:

• Single File – all selected contigs are saved in a single file.
• Multiple Files – each selected contig is saved as a separate file.

How can I see my DNA chromatograms?

In SeqMan Ultra, open the Alignment view, then click the plus sign to the left of a sequence name to display its chromatogram. Chromatograms are available only for ABI sequences.