Don't get species level assignment of blast results from custom reference library

User Questions
1

Hi,
When I do a remote blast to NCBI with NGS data, then import blast into MEGAN to assign taxonomy to reads, everything works fine, I get assignment down to species level. I have built a custom reference database and have done a local blast with the same NGS data against the custom database and when I import those results into MEGAN, I only get down to genus level taxonomic assignment. I have played with the LCA parameters to no avail. Perhaps my custom reference library is not formatted correctly for MEGAN to interpret the blast results fully and assign species?

The fasta file that I used to build the reference library so that I could do a local blast to it was formatted as shown below. For some species, I had more than one sequence and so needed to append the _1 _2 _3 etc to the name so all headers would be unique.

gnl_taxon_8098_Cottus_rhotheus_1
TTCTTG…
gnl_taxon_86918_Mylocheilus_caurinus_2
TCACACT…
gnl_taxon_8098_Cottus_hubbsi_3
TTTTC…
gnl_taxon_244772_Catostomus_columbianus_4
TACAC…

Any help would be appreciated. I am happy to provide more info if you let me know what would be useful.

Thanks.

2

The taxon Ids mentioned in your example are all genus level ids, so if MEGAN is capturing taxon_9098 for taxon determination, say, then it is not surprising that the assignment is at the genus level.
If you want species level assignments, then either replace the genus level ids by the appropriate species level ids, or write the species names into the header lines (with any confusing taxon ids and without underscores in the names).