Abstract: A method of generating a codebook includes obtaining a plurality of gene-identifying code words for the codebook. Each gene-identifying code word is represented by a sequence of N bits that correspond to a best match to a pixel data value identifying a gene. A plurality of negative control code words is generated, and each negative control code word is represented by a sequence of N bits. The negative control code words have an equal number of on-values. On-values of the plurality of negative control code words are evenly distributed across the N bits such that each ordinal position in the sequence of N bits has a same total number of on-bits from the plurality of negative control code words, and a Hamming distance between each negative control code word and each gene-identify code word is at least a distance threshold.

Abstract: A method of method of spatial transcriptomics includes receiving a plurality of images of a sample from an mFISH imaging system and generating a pixel word represented by a sequence of N intensity values. For each pixel, the pixel word is compared to a codebook and a closest matching code word of a plurality of code words is identified. Each code word is represented by a sequence of N bits. The plurality of code words include a plurality of gene-identifying code words and a plurality of negative control code words, the plurality of negative control code words have an equal number of on-values, and on-values of the plurality of negative control code words are evenly distributed across the N bits such that each ordinal position in the sequence of N bits has a same total number of on-bits from the plurality of negative control code words.

Abstract: Exemplary embodiments provide methods, mediums, and systems for generating a library of oligonucleotides for fluorescence in-situ hybridization transcriptomics probes. The illustrative techniques include several improvements, which may be utilized separately or together. These improvements include automatically iterating over a particular group of probe building actions while excluding other actions from the automatic iterations. This serves to reduce the amount of processing and memory resources required while significantly speeding up the process of building the library. Other improvements described simplify the input of genes of interest to be used to construct the probes and provide quality control capabilities. The described solution may be implemented in non-script-based instructions, which simplifies the input procedure, allows for the separation of data management and processing capabilities, and reduces the need for expert users to build the library.