hcapca is an R script for performing Hierarchical Clustering Analysis (HCA) and Principal Component Analysis (PCA) on LC-MS data. It was tested on the following operating systems:

• macOS 10.14.4
• Ubuntu 18.04.2 LTS
• Windows 7
• Windows 10

Install Docker Community Edition (CE) for your operating system. For older Mac and Windows systems, you will need to install Docker Toolbox instead.

Example data, R scripts, and config_file.yaml can be found by downloading this repo such that your directory structure looks like this:

base
  |--config_file.yaml
  |--hcapca.R
  |--accessory_functions.R
  |--app.R
  |--data
      |--example_feature_table.csv

You can use your own data instead of example data here. The format of each file must be like the example data.
Note: The base directory is where you unzip the data folder, R scripts, and config_file.yaml.

• You must have administrator access to install Docker or Docker Toolbox.
• You should increase the memory limits to allow the script to run. I recommend 4 GB of RAM and 2 cores to run the example data.
  • For Windows 7, open VirtualBox (installed as part of Docker Toolbox) from Start Menu as admin and stop the virtual machine default that is running. In settings for default, change the RAM and processor allocation.
  • For Linux, you don't need to do this since Docker has access to the entire system's resources.
  • For Windows 10 and macOS, open the preferences from the Docker app and increase resources as needed.
• You must enable shared folders:
  • In Windows 10, you need to enable shared folders in preferences. Right click on the Docker icon in the system tray > settings > shared drives > check appropriate drives > Apply
  • In Windows 7, as before, access the VirutalBox as admin > stop the default virtual machine > go to settings for the virtual machine > Shared Folders > Add as needed
• For Windows 7 please make a note of the IP address that is displayed when you first start the Docker Quickstart Terminal. Usually it is something like 192.168.99.100. This will be important in step 5 below.

Open a terminal, cd to the base directory and run:

docker run --rm \
 --tty \
 --interactive \
 --volume $(pwd):/srv/shiny-server/hcapca:rw \
 --workdir /srv/shiny-server/hcapca \
 schanana/hcapca:devel hcapca.R

Use the Powershell (not x86 or ISE, just Powershell) and type:

docker run --interactive `
  --tty `
  --rm `
  --volume //c/Users/username/path/to/base/directory:/srv/shiny-server/hcapca `
  --workdir /srv/shiny-server/hcapca `
  schanana/hcapca:devel hcapca.R

Be sure to replace /username/path/to/base/directory in the above command with the path to the base directory.

Use the Docker Quickstart Terminal, cd to the base directory and run:

 docker run --interactive \
   --tty \
   --rm \
   --volume $(pwd):/srv/shiny-server/hcapca:rw \
   --workdir /srv/shiny-server/hcapca \
   schanana/hcapca:devel hcapca.R

You can use devel in place of latest in the last argument for a more cutting edge version.

base
   |--output
         |--report.html
         |--hca
         |   |--lots_of.pdfs
         |
         |--pca
            |--names_with_underscores.html
            |--directories_with_the_same_names

If your config file has parameter output_pca as FALSE, then the pca folder will be empty. Do not worry, you can explore individual PCAs for each node in the next step.

In the base run:

docker run --rm \
   --interactive \
   --tty \
   --name hcapca \
   --detach \
   --volume $(pwd):/srv/shiny-server/hcapca:rw \
   --workdir /srv/shiny-server/hcapca/ \
   --publish 3838:3838 \
   schanana/hcapca:devel

Navigate to http://127.0.0.1:3838/hcapca to view your results in an interactive website!

Use the Powershell (not x86 or ISE, just Powershell) and type:

docker run --rm `
   --interactive `
   --tty `
   --name hcapca `
   --detach `
   --volume //c/Users/<username>/path/to/base/directory:/srv/shiny-server/hcapca:rw `
   --workdir /srv/shiny-server/hcapca `
   --publish 3838:3838 `
   schanana/hcapca:devel

As before, replace username/path/to/base/directory with the path to the base directory and navigate to http://127.0.0.1:3838/hcapca to view your results in an interactive website!

On Windows 7, use the Docker Quickstart Terminal and in the base directory run:

docker run --interactive \
  --tty \
  --rm \
  --detach \
  --name hcapca \
  --volume "$(pwd)":/srv/shiny-server/hcapca \
  --workdir /srv/shiny-server/hcapca \
  --publish 3838:3838 \
  schanana/hcapca:devel

Navigate to http://your.ip:3838/hcapca where you should replace your.ip with the ip shown when docker starts up - as mentioned in section #3 Housekeeping above.

Contains: Sample names
Format: One name per line; names can be any combination of letters and numbers. Duplicates will be removed.

A123  
B123  
C122
B455

Contains: m/z values separated by spaces all on one line. Keep them in the same order as the retention time and table data.

198.0390 758.3503 0.0000 395.1877 366.2404 ...

Contains: retention time values (in minutes or seconds) separated by spaces all on one line. Keep them in the same order as the m/z and table data.

12.3 8.0 7.3 2.02 12.3 ...

Contains: intensity values
Format: Space separated values; numbers only. Each line contains the values corresponding to the sample names from Analyses.dat above.

198.0390 758.3503 0.0000 395.1877 366.2404 ...
438.1456 378.1118 435.0968 525.2830 0.0000 ...
312.1296 349.1325 184.0433 0.0000 201.0702 ...
425.0835 0.0000 311.1384 371.2302 337.0677 ...

b0-15.pdf HCA of node b0 with 15 samples

b0_PC1-2_S.html PCA Scores plot of b0 for PC1 and PC2

• In some flavors of Windows, there may be an error in mounting a shared drive. If that happens, try the following:

  • Make sure the path is specified as //<drive_letter>/<path>
  • Make sure the shared drives are enabled and that particular path is shared
  • Make sure the terminal is running as administrator

• Docker abruptly stops running the script

  • Make sure to allocate sufficient RAM and processing power to Docker. Usually, if the virual OS cannot get more memory, it experiences an Out Of Memory (OOM) error and kills the offending process thereby exiting the container.

• For any issues please open an issue here on github or email me at [email protected]

Here is a link to wiki page which is still under construction.

This project is licensed under the GNU General Public License v3.0 - please see the LICENSE.md file for full license.

A huge thank you to Chris Thomas for helping bring this idea to fruition by being a wall to bounce off ideas. He does not have a github account so here is a link to a pubmed search with his publications. Another big thank you to @cecileane for pointing me in the right direction to generate a tree from a dataframe. Finally, a shoutout to @WiscEvan for helping figure out bits and pieces of code here and there, especially with the shiny UX.