Scalable Distributed Processing Software for Next-Generation in Situ Sequencing

A computer implemented method for processing in situ sequencing imaging data, the computer performing steps comprising:

• (a) receiving in situ sequencing imaging data;
• (b) applying configuration parameters to the in situ sequencing imaging data, wherein the configuration parameters comprise an encoding scheme, a codebook, image acquisition parameters, and sample metadata;
• (c) preprocessing imaging data by removing optical aberrations, subtracting background signal, performing deconvolution, filtering the images, and merging overlapping fields of view;
• (d) performing registration of imaging data by aligning selected common features in images taken at different timepoints and across different color channels at each time point;
• (e) segmenting images to determine locations of cell nuclei and cells;
• and
• (f) performing sequential analysis of images if the in situ sequencing imaging data is from sequential sequencing, wherein said performing sequential analysis of images comprises:
• i) measuring an intensity of a fluorescent signal for each segmented cell and nucleus, wherein the intensity of the fluorescence signal is proportional to the amount of a target nucleic acid, and ii) subtracting estimated carry-over signal from a previous sequencing round, or
• performing combinatorial analysis of images if the in situ sequencing imaging data is from combinatorial sequencing, wherein said performing sequential analysis of images comprises i) identifying each amplicon labeling a target nucleic acid from detection of barcodes, wherein invalid barcodes are removed according to their encoding scheme, ii) measuring a fluorescent signal of each amplicon at each timepoint to determine which color channel has the highest signal for each detected amplicon, closest code-book entry to a vector of signals, or clustering of pixel intensities;
• iii) iteratively matching signals across rounds of sequencing to membership in the code-book;
• iv) assigning a target identity to each detected amplicon, and v) calculating the number of each target nucleic acid present in each cell and nucleus.