title: Molecular Cartography Mouse 98 v3.0
status: Public on Oct 21 2022
contact.city: East Lansing
contact.first: Rance
contact.last: Nault
contact.email: naultran@msu.edu
contact.state: United States
contact.address: 1355 Bogue St. B403
contact.department: Department of Pharmacology and Toxicology
contact.country: USA
contact.institute: Michigan State University
organism: Mus musculus
manufacture_protocol: The probes for 98 genes were designed using Resolve’s proprietary design algorithm. Briefly, the probe-design was performed at the gene-level. For every targeted gene all full-length protein-coding transcript sequences from the ENSEMBL database were used as design targets if the isoform had the GENCODE annotation tag ‘basic’ (Frankish et al. 2019, Yates et al. 2020). To speed increase efficiency, the calculation of computationally expensive parts, especially the off-target searches, the selection of probe sequences was limited to sequences with high success rates. To filter highly repetitive regions, the abundance of k-mers was obtained from the background transcriptome using Jellyfish (Marçais & Kingsford 2011). Every target sequence was scanned once for all k-mers, and those regions with rare k-mers were preferred as seeds for full probe design. A probe candidate was generated by extending a seed sequence until a certain target stability was reached. A set of simple rules was applied to discard sequences that were found experimentally to cause problems. After these fast screens, every kept probe candidate was mapped to the background transcriptome using ThermonucleotideBLAST (Gans & Wolinsky 2008) and probes with stable off-target hits were discarded. Specific probes were then scored based on the number of on-target matches (isoforms), which were weighted by their associated APPRIS level (Rodriguez et al. 2017), favoring principal isoforms over others. A bonus was added if the binding-site was inside the protein-coding region. From the pool of accepted probes, the final set was composed by greedily picking the highest scoring probes.