10.5 The Contig Viewer

Contigs in Geneious Prime are viewed and edited in exactly the same way as alignments (see section 9.3 ). Features particularly relevant to contig assemblies are highlighted on Figure 10.6 and described below.

1. Consensus sequence

   The consensus sequence is displayed at the top of the assembly. This is the consensus of the reads only and does not include the reference sequence if one is present. The consensus settings can be found under the Display tab, and information about these settings can be found in section 9.5 . If the sequences in the contig have quality information attached we recommend selecting the Highest Quality consensus type. In most cases this removes the need for manually editing the contig because the consensus will be the base with the highest total quality at each position.

2. Coverage graph

   The coverage graph shows how many reads map at each position and can be useful for assessing the quality of your mapping. The Graphs tab enables you to show or hide the Coverage graph as well as enable other graphs such as the Identity or Sequence Logo graphs. See section 9.3.2 for a detailed description of these graphs. The data underlying these graphs can be exported in CSV format by clicking the Export option under the Graphs tab.

3. Reference sequence

   If you have run Map to Reference, the reference sequence is shown at the top of the assembly and is shaded yellow.

4. Color schemes

   The coloring of bases in the assembly can be selected from the dropdown Colors menu at the top of the General tab. Color schemes particularly useful for contig assemblies include the following:

   • For assemblies of Sanger chromatograms, the default color scheme is Base call quality. This assigns a shade of blue to each base based on its quality. Dark blue for confidence < 20, blue for 20 - 40 and light blue for > 40.
   • For assemblies of paired reads, the default color scheme is Paired Distance. With this color scheme paired reads are colored according to how close the actual separation between the reads is to their expected separation. Green indicates they are correct, yellow and blue indicate under or over their expected separation and red indicates the reads are incorrectly orientated. To configure the colors used, and the sensitivity for deciding if reads are close enough to their expected distance, click the Options link next to the color chooser. The status bar below the viewer will show the actual and expected separation between a particular pair of reads when you hover the mouse over one of the reads in the pair.
   • The Mapping Quality color scheme can be selected for reads mapped to a reference sequence. A mapping quality represents the confidence that the read has been mapped to the correct location. For a read with mapping quality Q, the probability that it has been incorrectly mapped is 10(-Q/10). For example, a read with a mapping quality score of 20 has a 1% chance of having been incorrectly mapped. Reads that could be mapped to multiple locations will have a maximum mapping quality score of 3, which indicates at least a 50% probability of the read mapping elsewhere. Mapping qualities have a maximum value of 254 for consistency with the SAM/BAM format. If a sequence has no mapping quality (i.e the document was produced in a version of Geneious prior to 8.1 or imported from a SAM/BAM file that didn?t have mapping quality) then it will be colored gray. Mapping quality for the sequence under the mouse is also displayed in the status bar. All mappers use heuristics to calculate mapping qualities. For unpaired reads, the Geneious mapper assigns a mapping quality of 20*(the number of additional mismatches in the second best location the read maps to). For paired reads the individual unpaired mapping qualities are calculated, but these are increased by up to 20 depending on how close the best pair is to the expected insert distance compared with the second best pair.
   • The By Direction color scheme allows you to quickly see whether reads are mapped in the forward or reverse orientation.
5. Advanced Layout settings

   The settings under the Advanced tab contain options for adjusting the layout of the contig.

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   Figure 10.7: Advanced layout settings for the contig view

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   Assemblies of short read data are normally viewed with the option Vertically compress contig enabled. This option puts the reads side by side where possible so that multiple reads can be viewed in the same row. Vertically compress contig cannot be enabled if Wrap contig is switched on. Wrap Contig will wrap the assembly to the screen width and is not recommended for large assemblies.

   Hide sites over x % gaps hides sites that have over the set percentage of gaps, so that indels introduced by sequencing errors do not interfere with the viewing of the alignment. This setting does not hide sites where the reference is a non-gap, and does not hide scaffolding gaps in de novo contigs. This setting can only be enabled when wrap contigs is switched off, and editing mode is switched off.

   Link Paired Reads: With this option on, pairs of reads will be laid out in the same row with a horizontal line connecting them. This option is only visible on contigs containing reads that have been paired before assembling - see section 10.2.1 . Reads separated by more than 3 times their expected distance are not linked by default unless the Link distant reads setting is turned on.

   The horizontal line between paired reads is colored according to how close the separation between the reads is to their expected separation. The coloring is the same as for Paired Distance coloring described above, where Green indicates they are correct, yellow and blue indicate under or over their expected separation and red indicates the reads are incorrectly orientated.

   If Link Paired Reads is off, it is still possible to view the connecting line between any pair of reads by mousing over one of the reads. To find the pair of a particular read, right click on the read and choose Go to paired mate.

Finding regions of low/high coverage

In addition to the coverage graph which gives you a quick overview of coverage, under the Annotate & Predict toolbar is the Find Low/High Coverage feature. This feature annotates all regions of low/high coverage which you can then navigate through using the little left and right arrows next to the coverage annotations in the controls on the right. You can set the threshold low/high coverage by either specifying an absolute number of sequences or a number of standard deviations from the mean coverage.

The find low/high coverage tool can also be used to record the minimum, mean, and maximum coverage of each annotation of a particular type on the reference sequence. To do this, in the Only Find In section of the options, turn on Annotations in reference sequence of type and choose Create annotations of same type on reference sequence.