Link Search Menu Expand Document

Great....yet another TMA dearray program. What does this one do?



Coreograph uses UNet (Ronneberger et al., 2015), a deep learning model, to identify complete/incomplete tissue cores on a tissue microarray, and export them individually for faster downstream processing. It has been trained on 9 TMA slides of different sizes and tissue types.

Training sets were acquired at 0.65 microns/pixel resolution and downsampled 1/32 times, or 2^5 times, to speed up performance. Once the center of each core has been identifed, active contours is used to generate a tissue mask of each core that can aid downstream single cell segmentation. A GPU is not required but will reduce computation time.

Prerequisitie input files:

-a fluorescence image of a tissue microarray where at least one channel is of DNA, ie. labelled with Hoechst or DAPI. The image will likely be a stitched images that spans multiple tiles.

Parameter list:

The Coreograph parameters described in this manual should be provided to mcmicro via the --core-opts flag, as shown in Parameter Reference examples.

  1. --downsampleFactor : how many times to downsample the raw image file. Default is 5 times to match the training data.
  2. --channel : which is the channel to feed into UNet and generate probabiltiy maps from. This is usually a DAPI channel.
  3. --buffer : the extra space around a core before cropping it. A value of 2 means there is twice the width of the core added as buffer around it. 2 is default.
  4. --outputChan : a range of channels to be exported. -1 is default and will export all channels (takes awhile). Select a single channel or a continuous range. –outputChan 0 10 will export channel 0 up to and including channel 10.

Output files:

  1. individual cores as tiff stacks with user-selectable channel ranges
  2. binary tissue masks (saved in the ‘mask’ subfolder)
  3. a TMA map showing the labels and outlines of each core for quality control purposes
  4. a text file listing the centroids of each core in the format: Y, X

map

Scenarios

NOTE: When using Coreograph on O2, the O2tma profile should be used!!

1. Alright, let’s get started!

When using MCMICRO, Coreograph does not require any additional input parameters to run. The DNA channel is assumed to be in the 1st channel.
--core-opts: <leave blank>

As one can see, each core is labelled with a single number implying that each core was found uniquely. Furthermore, each core has a thick white line to indicate the accuracy of segmenting each core. (Future versions will have a colored outlines for better visibility).

2. Well, my DNA channel is not in the 1st channel.

No problem! Specify --channel with the channel that it’s in. This is 0-indexing. So 1st channel is 0. If it’s in the 4th channel,
--core-opts: --channel 3

3. The cores aren’t being found properly.

Coreograph is trained on various core sizes ranging from 500 microns to 2 mm acquired at a pixel size of 0.65 microns per pixel and then downsampled 5 times. If your core size or image resolution are significantly different, you will need to either upsample or downsample a different number of times using --downSampleFactor. See below for examples:

3a) If your pixel size is 0.325 microns per pixel, then your pixel size is double the training data by a factor of 2 (0.65/0.325). You should downsample more times. Use 6 instead of 5.

map
--core-opts: --downsampleFactor 6

3b) If your pixel size is 1.3 microns per pixel, then your pixel size is half of the training data (0.65/1.3). Instead of downsampling by 5 times (default), you should downsample less. Try 4.

map
--core-opts: --downsampleFactor 4