10.2.1 Setting paired reads

To assemble paired read (or mate pair) data, prior to assembly you first need to tell Geneious the reads are paired. The assembler will then automatically used the paired data unless you turn off the advanced option to Use paired distances.

Paired reads can be set up either during the fastq import process, or by selecting the documents(s) containing the paired reads and going to Set Paired Reads from the Sequence menu. Depending on your data source, reads could be in parallel sets of sequences, or interlaced, so you need to select the appropriate format. Geneious will guess and select the appropriate option based on the data you have selected, so most of the time you can just use the default value for this. However, you must make sure you select the correct Relative Orientation for your data. Different sequencing technologies orientate their paired reads differently. All paired read data will have a known expected distance between each pair. It is important you set this to the correct value to achieve good results when assembling. If you don’t know what the relative orientation or expected distance is between the reads you should ask your sequencing data provider.

When you click ‘OK’, if you chose to pair by parallel lists of sequences a new document containing the paired reads will be created. If you do this during the fastq import process, Geneious will only import the paired read file and not the original unpaired files. If you chose to pair an interlaced list of sequences (or modify settings for some already paired data), the existing list of sequences will be modified to mark it as paired.

If you choose to split reads based on the presence of a linker sequence (e.g. for 454 data) the original sequences will be unmodified and the split reads will be created in a new document. The default behaviour is to ignore sequences shorter than 4 bp either side of the linker, but this can be customized from the Edit Linkers option in the paired reads options.

Polonator sequencing machine reads can be split using the Split each read in half option.