Designs third-generation HCR probes (HCRv3, split-probes) and attaches split initiator sequences.
- B1-5, Choi 2014 ACS Nano
- B7, B9-10, B13-15, B17, Y Wang 2020 BioRxiv
Please see user manual PDF file for a detailed guide.
Install BLAST from https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/. Use *.dmg file for Mac. Then check that R can find the installed BLAST. If it can't, add the BLAST installation path to R system path using Sys.setenv(PATH = paste(Sys.getenv("PATH"), path/to/blast/bin, sep = .Platform$path.sep)).
Sys.which("blastn") # should say sth like "/usr/local/ncbi/blast/bin/blastn"
For Linux only, install libpq-dev on your linux machine. Not required for MacOS.
sudo apt-get install libpq-dev
Install metablastr and dependencies (https://github.com/drostlab/metablastr) in R. Make sure your Mac has XCode installed in order to install devtools.
# if (!requireNamespace("BiocManager", quietly = TRUE))
# install.packages("BiocManager")
BiocManager::install(c("Biostrings", "GenomicFeatures", "GenomicRanges", "Rsamtools", "IRanges", "rtracklayer", "biomaRt"))
# install.packages("devtools")
devtools::install_github("HajkD/metablastr", build_vignettes = TRUE, dependencies = TRUE)
Decompress the BLAST database fasta file and copy/unzip to a desired directory. D.mel BLAST database based on ENSEMBL v99 is provided in this repo. Message me for Human and Mouse BLAST databases.
# cd to the repo directory
gzip -cd data/BLAST/Dmel_BLASTdb_ens99.fa.gz > /path/to/desired/directory
install.packages(c("tidyverse", "furrr", "patchwork", "valr"))
Follow the HCRv3_probe_design_workbook.qmd workflow. Input target sequence can be supplied as a FASTA file, or manually paste the sequence to target_raw variable. The desginer automatically removes empty characters and it is not case-sensitive. Either DNA or RNA sequence can be used as input.
See Zenodo publication for a more detailed user manual.
If ran successfully, there will be five outputs in a named output folder:
probes.txt : final probe sequences to order
probes.pdf : placing of probes along the target RNA sequence
details.csv : thermodynamics and other calculated values of the final probes
params.txt : parameters used for the design
rawblastoutput.csv : Raw blast output of candidate probes