ZFIN groups reagents that target a specific nucleotide sequence, Morpholinos (MOs), CRISPRs and TALENs together and calls them sequence targeting reagents (STR). When one of these reagents is published, ZFIN staff checks that the published sequence matches the genome and contacts authors if there are any questions about either the MO sequence or the target sequences of TALENs and CRISPRs. The most common issues are: sequences that aren’t reported in the paper or sequences that don’t match the current genome build. Once the sequences are checked they are added to ZFIN, and are incorporated into reagent specific BLAST databases (https://zfin.org/action/blast/blast ) and where possible mapped onto the genome.
To map the STR targets in the genome the sequences are analyzed using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) and mapped onto the genome for display on the ZFIN GBrowse map (http://zfin.org/gb2/gbrowse/zfin_ensembl/?label=knockdown_reagent). This mapping location is displayed on the individual data pages as thumbnails of the genomic region. If a sequence doesn’t match the genome sequence an image doesn’t appear on the reagent page. When a STR hits multiple places exactly the locations are all shown on the page as an example MO1-mir375-1,mir375-2 targets both mir375 genes (https://zfin.org/ZDB-MRPHLNO-071106-1). Many Morpholinos don’t match the genome because they were either targeted to an older version of the genome, or they cross a splice site so there isn’t an uninterrupted genome match. Multiple equivalent genome matches occur for several reasons; in some cases the reagents were designed to hit multiple genes in the same family but many other cases exist where multiple target sites seems to have been unintentional.
We always recommend double checking the target of a knockdown reagent. If there is no image on a page it is a sign that reagent might not target as expected.