This is a padlock design pipeline for multiplexed assay with multiple probes per target in cDNA-based expression profiling Mats Nilsson Lab, Stockholm University Xiaoyan, 2018 How to install and run: Install blast+, add to path Install clustalW, add to path Install Python 3, add to path Add folder Scripts in Python installation folder to path Open command window, type: pip install numpy From ftp.ncbi.nlm.nih.gov download refseq mouse and human mRNA sequences Download this repository and unzip Modify config.py file to specify where you have saved mRNA sequence files Modify config.py to specify maximum number of parallel threads for MSA (multiple sequence alignment) and blast search Double click probedesign.py to run The software allows the following input files: 1. A text file with a list of gene acronyms. Only works for humand and mouse transcriptomes. One row per gene. 2. A csv file with gene acronyms and corresponding linker and barcode sequences. One row per gene. No header in the file. 3. A text file with target sequences in fasta format. Other parameters: Species: the database against which the sequence specificity should be checked. This will also be the database to look for gene acronyms if input fasta sequences are not provided. Padlock arm length: the length of each target arm of a padlock probe. The final target sequence will be twice this length. Tm: lower and upper limit of melting temperatures, in a reaction containig 0.1 uM probe, 0.075 M monovalent salt, 0.01 M bivalent salt and 20% formamide. Fixed parameters: For specificity check, in order to say a target sequence is specific enough, it cannot have any sequence in the database with more than 50% of sequence coverage, covering ligation site +- 5 nucleotides, and 80% of homology.