Once you have created a SeqMan Pro assembly, the editing strategies to consider include:
•Review the frequency of conflicts from the Alignment View.
•If you find regions with high frequencies of conflicts (i.e. differences between constituent reads at the same consensus position), you may view the underlying trace data to try to resolve any conflicts between constituent sequences. Note, however, that even when there are conflicts in your assemblies, SeqMan Pro’s trace quality evaluation system calls the consensus sequence based on the relative quality of the underlying traces when trace data are available. In most instances, this saves you the time involved in reviewing individual traces.
•Check for ambiguities in the consensus sequence by performing a Conflict search of the consensus sequence. If your consensus is based on trace evidence, you may be able to resolve ambiguities by lowering the Evidence Percentage value. Note that the lower the Evidence Percentage value, the more likely the software will call one of the four possible bases rather than an ambiguity, even when there is some evidence for an alternative base.
•Check for conflicts in the constituent sequences by performing a Conflict search of All Sequences in Contig. If your consensus is based on trace evidence, you will typically observe that the bases in the constituent sequences that conflict with the consensus are of lower quality than other bases in that position.
•If you are displaying two different consensus sequences, check differences between them by selecting Edit > Find selecting Consensus Difference. The Trace Evidence consensus method is more accurate than majority consensus methods, and you can see this feature in action if you display both Trace Evidence and Majority consensus sequences at the same time.
•If you decide to edit your assemblies, you may use the translation of the sequence to guide your editing decisions. For example, if the region of interest originates from what you suspect is an open reading frame; an apparent stop might signify an error in one or more constituent reads.
•If you are using .abi trace files, you may want to check instrument conditions that could have affected the outcome of a run. For example, a low temperature during electrophoresis can cause sequence artifacts. To review data collection conditions, select Sequence > Show Seq Info.
•To search for homologs that may help to confirm your assembly, you may select Net Search > BLAST Search. A related sequence in the BLAST database may help to resolve an ambiguous base.
•If an initial assembly yields multiple contigs, you may be able to join some or all contigs without the need for additional sequence data. Start by restoring quality-trimmed bases (i.e., bases with quality scores below your threshold) for the constituent sequences located on either end of the contigs. (See Extending Contig Ends for an automated method or Trimming and Restoring Sequence Ends Manually for a manual method.) Then, try merging the longer contigs by selecting Contig > Align Contigs.
•Another method for reducing the number of contigs involved reassembling the contigs using lower stringency conditions. Be aware, however, that this method may lead to false joins.