Viewing And Editing Contigs
As with the reads, contigs are each assigned a bin based on various quality criteria. This gives an initial measure of quality. You may decide to assume High bin contigs don't need to be looked at but Medium bin contigs should be examined in detail.
By default Geneious uses a Highest Quality consensus which rarely generates ambiguities because the highest quality base call is used automatically. However, ambiguities are generated in situations where the quality of conflicting bases are similar.
The standard procedure for checking disagreements in a contig is as follows:
- Select a contig and an overview of the contig will be displayed. Disagreements are shown as small black marks on the sequences and the trimmed regions can be seen.
- If this is the first contig you have looked at, turn on Allow Editing in the Contig View toolbar.
- To jump to the first disagreement in the contig press Ctrl+D (or Command+D on Mac OS). If this is the first contig you have looked at then you should zoom in to a level you are comfortable with shortly after going to the disagreement and Geneious will remember this for the next contig.
- After pressing Ctrl+D Geneious will select the base call which is in disagreement with the consensus (the lower quality base). If you agree with the call that Geneious has made then you can jump to the next disagreement because the call has already been made.
- If you disagree with the call or if an ambiguity is in the consensus, you can resolve the conflict by editing either of the reads or by editing the consensus which is a shortcut for changing all base calls at that position.
- Continue jumping through the disagreements until you have looked at all of them then save the contig and repeat for the next contig.
If you decide that some contigs are not good enough despite assembling correctly then you can mark these as failed at this point. Simply select the contigs that have failed and go to Mark as Failed in LIMS. It is a good idea to move the failed contigs to a new subfolder (eg. named "fail") so they don't interfere.