Input Data Preparation

The ExSequence pipeline uses a particular directory structure which by default consists of the following folders:

• 0_raw
• 1_deconvolution
• 2_color-correction
• 3_normalization
• 4_registration
• 5_puncta-extraction
• 6_base-calling

There is a folder per stage where the outputs of the stage are stored. It is important to note that performing deconvolution on input images is optional and not done by ExSequence. In our experiments, deconvolution did not improve results of the pipeline but results may vary by dataset. In addition to the folders for the stages, there are folders for storing logs and analysis images as well as a temporary directory. A temporary directory is necessary to store intermediate processing files in some of the stages. All of these folders can be set by parameters file stored in the ExSequence directory or alternatively, if using this directory structure, can just specify an output folder containing the above list of folders.

Apart from the directory structure, the input filename names must have the format:

• basename_roundXXX_chYY.ext.

That is a base filename followed by a underscore and round with a 3 digit number with leading zeroes (i.e round 1 would have basename_round001 and round 10 will have basename_round010), followed by another underscore and the channel string, and finally the file extension.

As with other aspects of ExSequence, the exact base name and channel strings can be customized in a parameters file or via command line options. By default, the channel strings are as listed with ch followed 2 digits with leading zeroes. It is important to note that the number of color channels must be exactly 4. Finally the input file extensions and intermediate image files can either be hdf5 or tiff.