Trimming sequences in Geneious is a little bit different from how trimming has traditionally worked. Instead of permanently deleting trimmed regions from the sequences, Geneious graphically annotates the trimmed regions meaning that the information is retained through downstream analysis and can be adjusted at any time. It is not necessary to delete trimmed annotations in Geneious, because assembly and other analyses automatically take the trims in to account.
Select all of the sequences you are going to assemble and select Sequence > Trim Ends... from the menu.
TIP: You can add Trim Ends to the main toolbar by right-clicking on the toolbar and turning on Trim Ends.
For most applications, the default of Error Probability Limit: 0.05 is a good start. This option works by trimming the sequence to find the longest possible untrimmed region which has an overall error probability below this number. Decrease the limit to trim more aggressively.
Other options include screening for vectors which uses a clone of NCBI's VecScreen tool, screening for primers (see section below) and basic limiting options such as minimum amount to trim.
When you click OK to run trimming it will add annotations to each of the sequences which correspond to the trimmed regions. You can flick through all of the trimming results in the Sequence View before saving the changes. If there are reads which are obviously not good enough quality for assembly then you can mark these as failed in the LIMS (as detailed later), but it is easier to let the assembly report pick these out for you (also described later).
Primer trimming in Geneious uses a custom Smith-Waterman search to locate primer matches in sequences.
Before using the primer screening feature you will need to establish a database of primers in Geneious. This is done by creating oligo type sequences which can be stored anywhere in your local folders (or you can create a specific "Primer" folder if you want to store them all in one place.
Oligo sequences are created in one of the following ways:
- Extract a primer/probe annotation from a sequence
- Select “Sequence” → “New Sequence” from the menu and choose Primer or Probe as the type of the new sequence
- Select one or more existing primer sequences (these may be imported from a file, e.g. a fasta file) then click “Primers” → “Primer Characteristics” to transform them into oligo type sequences
Once you have created oligo sequences for all of your primers, the Screen for Primers option will search for matches with any of them.
Sequences can be retrimmed using different parameters at any stage. To do this just select the sequences for retrimming and follow the steps above for trimming. The only difference is you should select the "Annotate new trimmed regions" option to have the new trims replace the old one.
When a sequence is retrimmed it currently stores the history of trims that were used in the Notes tab for each sequence.
Sequences can also be manually trimmed by selecting a region at the end of a sequence in the Sequence View then clicking Annotate and choosing Trimmed for the annotation type. If a sequence has more than one trimmed annotation at its ends then the largest trimmed annotation will be used. You can also manually edit any trimmed region by clicking and dragging either end of the trim annotation in the sequence view.