Microbial Metabolomics Research Center
Important: This repository is a template and training tool! Each dataset is different; while this pipeline handles many of the scenarios that arise within the data wrangling process, the user will need to adjust the parameters for their specific input data. This also applies to the example data from the Ingalls Drive. The example csvs are a handful of possible combinations of run type/instrument/software, but there is not an example sheet for every case.
The Targeted Pipeline is a combined process for importing, tidying, Quality Controlling, BMISing (see below for more details on this step), and quantifying (where possible) targeted metabolomics data produced by the Ingalls Lab.
It is run by a “Control.Rmd” markdown script that moves through the pipeline step by step, accounting for two mass spectrometer instrument types (TQS or QE), run type (HILIC/Reverse phase), ion mode (positive/negative), and metabolomics processing software (MS-DIAL or Skyline).
The Control.Rmd script contains detailed instructions, and the user must read carefully through each section description while progressing through the pipeline.
The pipeline contains four major sections:
- Import and cleaning/rearranging of data.
- Quality control using user-defined parameters.
- Applying a Best-Matched Internal Standard (B-MIS).
- Quantifying peak area to umol/vial when possible.
Below is a list of packages and external sofwares that this pipeline utilizes. Please ensure you have the package(s) installed and have access to the tools listed below.
| Name | Description |
|---|---|
tidyverse |
Data science package in R. |
data.table |
Provides an enhanced version of data.frame. |
anytime |
General purpose date converter. This package helps the user track analysis reruns and produce unique identifiers for files. |
rlist |
Non-tabular data manipulation toolbox. |
BMIS |
R script for batch-specific normalization of raw peak areas. Matches measured metabolites with isotope-labeled internal standards that behave similarly during the analysis, nicknamed Best-Matched Internal Standard or B-MIS. This pipeline contains a modified script taken from the original repository. Published paper here: https://pubs.acs.org/doi/10.1021/acs.analchem.7b04400 |
Example Data |
Pretidied data from the Ingalls Lab for learning purposes. If you do not have access to this drive, please contact the owners of this repository. |
The first few chunks of the Control.Rmd script will create empty folders for different types of data (raw, processed, extra, intermediate). Some of this data will be produced by the script (processed, intermediate) and some needs to be moved into those folders before proceeding. Because this is a template script, the data has been modified for easy input, which will NEVER be the case in a real run.
All the necessary example data can be downloaded here: https://drive.google.com/drive/folders/1E-B4dyDTkwOfupHJK0uL8BgKj586OU31
If you do not have access to this drive, please contact the owners of this repository.
These parameters will change depending on your data and level of stringency. Below are the “default” values.
area.min: The minimum area of an integrated peak to be considered a real peak. HILIC - 1000, Reverse Phase - 5000.
RT.flex: Flexibility of the deviation of the sample retention time from the appropriate standard. HILIC +/- 0.4 min, Reverse Phase +/- 0.2 min.
blk.thresh: Cutoff ratio value of sample area to a blank reference sample. HILIC +/-0.3, Reverse Phase +/- 0.2.
SN.min: Cutoff ratio value for Signal to Noise. HILIC +/- 4, Reverse Phase +/- 4.
height.min: Minimum height for a peak. 1000, HILIC and Reverse Phase.
height.max: Maximum height for a peak. 1.0e8, HILIC and Reverse Phase.
IR.flex: The TQS produces quantitative and secondary trace values. Find the Ion Ratio by dividing the area of the quantitative trace by the area of the secondary trace for standards, and use these ratios to create a reference table. Deviations from this reference table that are larger than the user-defined values will be flagged. 0.3, HILIC and Reverse Phase.
Pull requests are welcome. For major changes, please open an issue first.