Now that you have completed Part B, step 1, this section will show how to review the putative non-synonymous variants.

  1. Click on the Contig Pos column header to sort variants in ascending order of their positions in the alignment.
  1. Look at the Impact column for the variant in the top row, and note that a non-synonymous mutation is predicted.
  1. Double-click on the row to launch the Alignment View with the variant highlighted.

The Alignment View displays the consensus sequence, and the alignment of all sequences making up a contig, at the nucleotide level. The consensus sequence for the selected contig is displayed at the top of the window, immediately beneath the scale. Constituent sequences are displayed in the bottom section.

  1. Click a few bases to one side of variant position 63,858 to temporarily clear the selection. This will allow for a better view of the color-coding used by SeqMan Pro.
  1. In the top left of the Alignment View, click the triangle to the left of “Consensus” to display the reference sequence on the top row and any disagreements in the second row.

Note that the Reference base at the position of interest is C, while the Majority of samples contain T.

The colors of bases in the aligned reads are determined via the Colors section of Project > Parameters > Variant Color. By default, the color for putative variants (variants that have been neither confirmed nor rejected) is blue.

  1. To view the variant in the Strategy View, select Contig > Strategy View. The Strategy View graphically summarizes the position and orientation of every constituent sequence in the selected contig.
  1. Zoom in as far as possible by repeatedly clicking the Zoom In tool ().
  1. Return to the variant position by selecting Edit > Go To Position. Type 63858 into the box, select the uppermost radio button, and then press OK.

In the lower part of the window, observe the green arrows. Arrows are used to graphically represent the individual sequences in the contig. The point of the arrowhead represents the 3’ end and the tail represents the 5’ end. The green coloration of arrows in this region denotes consistent, correctly-specified paired reads.

  1. To display areas with conflicts, check the Conflicts box in the top left corner of the Strategy View. Calculation of conflicts will take approximately 5-10 minutes.

The Strategy View header now displays useful information for evaluating the variant.

  • Coverage Threshold – Represents the coverage within the assembly as it compares to the threshold parameters defined in Project > Parameters > Strategy Viewing & Coverage. In this example, the thick green bar denotes above-threshold coverage along this portion of the consensus.
  • Conflicts – The black histogram represents how well the constituent sequences agree with the consensus. Note that there is a conflict area centered around the putative variant.
  • Depth of Coverage – Represents the depth of sequences throughout the assembly. The numbers to the left of the graph indicate the actual number of sequences represented. In this example, the Depth of Coverage is excellent, as denoted by the thickness of the green area.
  • Pair Consistency – Displays a summary of consistent (green) and inconsistent (red) paired end sequence data content. The green bar indicates excellent pair consistency.
  • Features – The putative variant is located within a labeled gene called mraZ.

Proceed to 3. Adding variants to a custom database.

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