Assembly of all pairs of reads is done in one step using "assemble by name". To do this, select all of the reads you are going to assemble then click Assembly in the toolbar. Below is a step-by-step description of the assembly options, along with recommended settings.
- Firstly you can select a reference sequence. Make sure you have selected the reference sequence along with your reads, and select it from the dropdown at the top of the assembly dialog.
- Next are the assemble by name options which tell Geneious which part of the names each pair has in common.
- The recommended assembly method is 'High Sensitivity' or "Highest Sensitivity' as we are dealing with small assemblies (so there will not be much speed difference between settings). It is also possible to choose 'Custom Sensitivity', and choose your own parameters (for example, minimum overlap).
- If you have already trimmed your sequences, select "Use existing trim regions". Otherwise you can set your trimming options here to trim with assembly in a one-step process.
- "Save assembly report" and "Save results in a new subfolder" should both be turned on.
After clicking OK, a new subfolder called "Assemblies" will be created and contigs will be added to it as the operation runs. When the operation is finished an Assembly Report document will also be added to the folder (see below). If you decide to run another assembly with different settings, Geneious will create a new subfolder so the results are kept separate.
The Assembly Report
The assembly report is a record of which reads were assembled successfully and which failed. This can be printed out if desired.
The assembly report is also a great tool for identifying the first sequencing failures. Any sequences which totally failed to assemble can be easily selected and then marked as failed in the LIMS. At the bottom of the report there is a Select All link next to the "All Unassembled Fragments" heading. Click this to select all of the reads which failed to assembly then you can easily use Mark as Failed in LIMS.