10.2.5 Removing chimeric reads

To remove chimeric reads from NGS datasets, select the sequence list containing your reads and go to Sequence Remove Chimeric Reads. This runs UCHIME by Robert Edgar and is typically used to remove PCR chimeras from amplicon sequencing (e.g. 16S, ITS). The public domain version of UCHIME is provided with Geneious. If you would prefer to detect chimeric sequences using USEARCH, which contains a much faster version of the UCHIME algorithm, you can optionally specify a USEARCH executable instead.

Geneious supports reference mode only, and you must supply the reference database yourself. This may be either a nucleotide sequence list or a nucleotide alignment in your Geneious database. Information about common reference databases for 16S rRNA or fungal ITS sequences is available, along with links to download locations, on the Geneious knowledge base. When you have imported your preferred database into Geneious, choose this document as the Reference Database.

If your query sequences are paired, you may need to run Merge paired reads before chimera detection. When a query sequence list with reads set as paired is selected, Geneious will always consider both members of a pair to be chimeric if either is identified as such. Note that UCHIME does not recognize paired reads, therefore by default Geneious will concatenate paired reads and submit each pair to UCHIME as a single sequence. This should generally be appropriate for reads that are separated by small gaps. To override this setting, you can check Run paired reads separately under More Options.

The following options are available for configuration within Geneious should you wish to optimize the settings for your data. The default settings in Geneious are consistent with the UCHIME defaults.

Installing USEARCH on Geneious Server

To run Remove Chimeric Reads... on Geneious Server, USEARCH will first need to be installed on the cluster. If you are not the Geneious Server administrator you will need to refer the appropriate person to these instructions.

First, download USEARCH, then install it on the Geneious Server cluster such that “usearch” is found and executable. You can do so in one of the following ways:

Note that the public domain verison of UCHIME (bundled with Geneious) is not supported on Geneious Server.