Editing a chromatogram

DNA chromatograms often contain regions where the base "call" is ambiguous. This is common at the very start of a DNA sequence read as it is difficult to resolve very small DNA fragments. In addition to identifying ambiguous bases, it is necessary to remove primer sequences since PCR primers are synthetic pieces of DNA and should not be included in the analysis of sequence data.

Open and edit each of the chromatogram documents. You can open the first sequence by clicking here.

Click on the Allow Editing button in the "Sequence View" - this will enable you to edit the sequences.

Remove the sequence that corresponds to the primer binding sites (the primers are included as selectable annotations if you cannot find the sequences) along with any sequence after this. Also, remove the poor quality data at the beginning of the sequence reads - this usually represents the first 15-20 bases of the sequence. Remember to Save your changes.

Now edit the second sequence in the same way. You can open it by clicking here.

Assemble your sequences

One of the benefits of sequencing PCR products in both directions is that the majority of base calls can be "checked" against the read on the other strand. Select both of the edited chromatogram files and choose De novo assemble... under the Align/Assemble button. If necessary, click Restore Defaults then click OK.

Once the assembly is complete, you should click "Consensus" in the sequence view options panel so that the consensus sequence is shown. Make any edits you think are necessary (remember to click Allow Editing to put Geneious into its sequence editing mode) and Save the changes you have made. If you need to delete or insert a base you must select the same position in all of the sequences so they remain correctly aligned.

Question 2: How many bases were edited in your alignment? - Give a brief justification as to why these edits were required.
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Once you have edited the alignment, extract the consensus sequence to a new file. In the alignment window, click on "Consensus" to the left of the alignment view and then click on the Extract icon. Geneious will open a window where you can set the name to be "Consensus bear bile".

Exercise 3: BLASTing your unknown sequence
Exercise 4: Build a phylogenetic tree