Saturday, June 4, 2011

Geneious Part II

I'm writing a short introduction to Geneious for use in our lab and I thought I could put a bit of it here:

Importing files
Sequences can be imported to a folder using File>Import. The Geneious autodetect feature is pretty accurate and supports .ab1 files from our sequencer, or nexus/phylip/fasta if you have previously editied sequences. To import a Sequencher project, you have to export the contigs from Sequencher in CAF format and Geneious will import these as contig assemblies with chromatograms.

Dealing with chromatograms
Geneious can assemble contigs of the forward and reverse traces. Simply select all the files you want to assemble, click 'Assembly' and choose either 'Assemble to reference' (e.g. for assembling primers to a sequence) or 'Assemble by name' (for matching forward and reverse sequences) and choose if you want Geneious to trim you sequences or not (I leave this ticked as it works really well).

Geneious will give you a report of which chromatogram traces assembled and which didn't. You can elect to save this list by checking the box next to 'Save assembly report' in the previous step.

You can now edit the contigs.
The piece with the red underline is the piece Geneious has auto-trimmed due to low quality. The chromatogram is still there for this piece, but it isn't included in the consensus sequence (at the top) and won't be included when we export the sequence for alignment. If you want the chromatograms to be bigger, you can click and drag the peaks upwards, or to make it take up more of the screen, change the number next to 'chromatogram' in the Graphs menu. A word of warning: the default colours of the peaks are different to Sequencher, but can be changed if you like by clicking 'edit' next to the colours dropdown box on the right:

When you make a change in the chromatogram view, Geneious puts a coloured bar under the changed base. Hovering the mouse over this tells you what change you made and when. This is a brilliant feature, very useful when we are editing our alignments. When you have finished, save and close the editing window to return to Geneious. 

When you have edited your sequences, select them all and hit 'Alignment.'


Choose 'create alignment of consensus sequences' to use your edited sequences, ignoring the trimmed bits. You now have an alignment file, which you can open and check to remove primers etc.

If you want to check a sequence, click the little arrow to the left of the sequence name and Geneious takes you straight to the contig (in the main window). As before,your editing history for the sequences in the alignment is visible as bars under the sequence. When done, you can re-align the sequences by selecting the alignment, hitting 'Alignment' on the top bar and selecting the method you want to use (Clustal, Geneious's own algorithm, the MAFFT plugin etc.). To install plugins, go to Tools>Plugins.

To build a tree from your alignment, select it and hit 'Tree' in the top bar. 
Plugins are available for MrBayes, PhyML and PAUP*, provided you have the executable for these and now the path to them. The full choice of options, model selections, partitions etc. can be typed in. The trees can be opened and viewed in Geneious.

To export any file (sequence, alignment, tree) File>Export and select a file format (nexus, fasta, phylip, genbank flat etc.).


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