Note: For Customer Support contact information, and for a link to the most up-to-date version of this help, please see Before You Begin.
SeqMan Pro is both a sequence assembler and editor for small scale sequence assemblies (plasmids, PCR products, etc.), and a viewer for all assemblies, including BAM alignment file based projects. For larger sequencing projects and those using next-gen data, DNASTAR’s SeqMan NGen is used for the assembly process, while SeqMan Pro is used to view and analyze the finished assembly.
Note that SeqMan NGen produces an assembly project containing BAM alignment files for each of the contigs, BAM index files, template files, variant files and coverage files. A BAM-based project may be gapped or ungapped, but always contains purely BAM contigs. BAM alignment files are not editable. Because of this, some SeqMan Pro procedures cannot be followed with BAM-based projects. Topics and commands that are not applicable to BAM-based projects are clearly marked in this manual.
Using SeqMan Pro to View and Analyze Assemblies:
As a viewer, SeqMan Pro allows you to open assemblies from SeqMan NGen (including multi-sample MID-tagged 454-based assemblies). Within SeqMan Pro, you can analyze the consensus, features, and other components using a wide range of views and reports.
Assembly information is summarized in SeqMan Pro’s tabular reports and graphical views. The Strategy View graphically summarizes the position and orientation of every constituent sequence in a contig or contig scaffold and allows you to easily assess the coverage in your assembly. If coverage seems unsatisfactory, you can add more sequences and reassemble, or you can use the primer walking feature to drive the closure of gaps or to fill in low coverage areas. The Alignment View gives you a more detailed picture of the assembly, and allows you to edit constituent sequences, override the called consensus, review underlying trace or flowgram data, add new features, restore previously trimmed data, and adjust alignments. You may also evaluate putative variants identified by SeqMan Pro. SeqMan Pro’s variant calling algorithm makes variant calls with exceptional accuracy. You can also force contigs to join, or split a contig into two or more segments. Once you are satisfied with your assembly, you may save your project, export the data, or merge contigs with those imported from previous assembly projects.
Using SeqMan Pro as an Assembler and Editor:
SeqMan Pro allows editing of SeqMan Pro (.sqd) and Phrap (.ace) assemblies and can also perform a statistical calculation of their variants. By contrast, SeqMan Pro enables you to assemble fragment data from small-scale sequencing projects up to tens of thousands of fragments. Most often, SeqMan Pro is used for Sanger based sequencing of PCR products, plasmids and small viruses. We recommend using SeqMan NGen, rather than SeqMan Pro, for most other assemblies, including: larger viruses, bacteria, eukaryotes with genomes from 100 MB to over 1 GB, human exomes, transcriptomes and metagenomic projects.
SeqMan Pro lets you assemble sequences from numerous file formats, including trace data from automated sequencers, DNA Multi-seq (.mseq) files with features, and data output created using 454™ technology (though we recommend using SeqMan NGen to assemble next-gen data). SeqMan Pro removes unreliable data, including poor quality ends, sub-minimal length reads, and vector and contamination host sequences in a single pass, then assembles the trimmed data and calls the consensus.
SeqMan Pro provides two different assembly methods: the Classic assembler, and the Pro assembler. In general, the Pro assembler should be used when your data: 1) are medium in size; 2) contain repeated sequences; 3) have noisy ends; or 4) are being used for variant analysis. The Classic assembler should be used when: 1) your data do not contain repeated sequences; 2) you do not use vector trimming; or 3) you want to reproduce an assembly made from a previous version of SeqMan Pro. As with all SeqMan Pro parameters, you may select one of these assemblers as your default assembly method. You may also switch between the Pro and Classic assemblers with each SeqMan Pro project.
For projects that utilize trace data from automated sequencers, SeqMan Pro includes DNASTAR’s unique Trace Quality Evaluation system. This system evaluates the quality of the underlying trace data, and then generates the most accurate consensus sequence possible. This method is so effective that it reduces the depth of coverage needed for accurate sequence determination, yielding spectacular savings in time and effort.
Before assembling the sequences, be sure to review SeqMan Pro’s preassembly options. These options include trimming poor-quality data manually or automatically, removing specific vector or contaminant sequences, and identifying repetitive sequences so that they will be added last during assembly. You may also adjust a wide variety of parameters for the assembly process, including which assembly method is used, as mentioned above. Once you have selected the options you want, click the Assemble button to activate all of the trimming, assembly and consensus calling options you selected. If you prefer to assemble certain groups of sequences separately, use the Assemble in Groups option.