Implement SeqFISH SpotFinding

[ambrosejcarr]

SeqFISH carries out spot finding using the following approach:

The potential intron signals were then found by finding local maxima in the image above a predetermined pixel threshold in the registered images. Once all potential points in all channels of all hybridizations were obtained, dots were matched to potential barcode partners in all other channels of all other hybridizations using a 2.45 pixel search radius to find symmetric nearest neighbors in 3D. Point combinations that constructed only a single barcode were immediately matched to the on-target barcode set. For points that matched to construct multiple barcodes, first the point sets were filtered by calculating the residual spatial distance of each potential barcode point set and only the point sets giving the minimum residuals were used to match to a barcode. If multiple barcodes were still possible, the point was matched to its closest on-target barcode with a hamming distance of 1. If multiple on target barcodes were still possible, then the point was dropped from the analysis as an ambiguous barcode. This procedure was repeated using each hybridization as a seed for barcode finding and only barcodes that were called similarly in at least 4 out of 5 rounds were used in the analysis. The number of each barcode was then counted in each of the assigned cell volumes and transcript numbers were as- signed based on the number of on-target barcodes present in the cell volume. All image processing and image analysis code can be obtained upon request.

Reference

Starfish's LocalSearchBlobDetector implements the local matching, but does not incorporate decoding, or consensus matching across anchor rounds.

Starfish should implement the consensus matching required by the SeqFISH spot finder.

[ambrosejcarr]

Starfish could process only very small SeqFISH fields of view using a per-fov processing model (~300x300px). I am going to pilot a per-round processing workflow using SeqFISH and open some issues detailing my proposed data flow for a coded assay that is processed per round.