How to decide what BLAST settings to use when searching for functional genes in a metagenome

Question

[crystal4]

I have several lists of ORFs from metagenomic samples. I'm looking for specific genes by BLASTing the ORFs against databases of genes with known functions (for example, a database of nirK genes). I am having trouble figuring what values I should use for BLAST parameters such as identity, coverage, and word size. I know there probably isn't an exact answer, but are there any guidelines or papers dealing with this topic?

Answer 1

[Mensur Dlakic]

It depends on what you are trying to achieve. Generally speaking, E-value will be more informative than the parameters you listed. If you are looking for orthologs, E <= 1e^-20 is probably a good starting point, along with high coverage. If you want to identify paralogs, or in general more distantly related proteins, E <= 1e^-5 should be a good cutoff. In the latter case you are not necessarily expecting either high identity or high coverage.

• https://academic.oup.com/nar/article/47/D1/D807/5160989
• https://www.nature.com/articles/nmeth.3830
• https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-4-35