Makeblastdb can spit out warning notices when a higher count of N ambiguous nucleotides occurs in the first row of fasta data, but goes ahead and completes the blast database. "Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: First data line in seq is about 45% ambiguous nucleotides (shouldn't be over 40%)" At moment the NCBI Blast+ wrappers (toolshed.g2.bx.psu.edu/repos/devteam/ncbi_blast_plus/ncbi_makeblastdb/0.1.01) are saying "yup those are errors", but i think they should be downgraded to just Notices? If the tool is part of a workflow it fails - on db's like RDP RNA this is quite a nuisance. Anyone else run into the same thing? d. Also see: http://www.acgt.me/blog/2014/5/15/fun-with-an-error-message-from-ncbi-blast Hsiao lab, BC Public Health Microbiology & Reference Laboratory, BC Centre for Disease Control 655 West 12th Avenue, Vancouver, British Columbia, V5Z 4R4 Canada