SeqMan NGen calculates the consensus sequence for a de novo assembly. After opening the project in SeqMan Ultra, you can elect to change the consensus calling parameters so that SeqMan Ultra can recompute the consensus. The Consensus dialog lets you choose the method for calling the consensus sequence.

To access the dialog, use Project > Project Parameters. If the Consensus section is not already active, click on the word Consensus on the left.

In the Call by drop-down menu, choose between Majority and Trace evidence. In the Minimum box, enter an integer as described below.

  • Trace Evidence directly reflects the quality of peaks in the trace data and is the most accurate method for generating consensus sequences for contigs. It is strongly recommended you use Trace Evidence for consensus calling whenever trace data are available. Sharp, well-defined peaks are assigned higher scores than less well-defined peaks.
  • Majority should be chosen when trace data are not available. If you select Majority, use the Minimum box to enter the minimum percentage of identical residues at each position required to identify the consensus base at this position. Ambiguous bases in the consensus denote a column of bases whose constituents do not meet this threshold. Ambiguous codes are assigned in these cases.

In the Minimum majority percentage box, enter the minimum percentage of a called base in a column to consider it the majority. The default is 75.

Enter a value for the Heterozygous peak threshold, used for called the secondary peak at heterozygous positions. With trace data, diploid heterozygous positions show two trace peaks at the same position. However, the secondary peak must be at least 50% the “size” of the primary peak to be considered a heterozygous variant. Trace data also has some background noise, so adjusting the Heterozygous peak threshold value is often necessary to get accurate variant calling in trace data.

Click OK.

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