TITLE: BAF_Protocol_011 Metabolomics: Database Search MS-DIAL and Analysis using Metaboanalyst 6.0
AUTHORS: Nicholas Sherman

[DESCRIPTION]
These steps represent a starting point for analysis of metabolomics data. Other data analysis and/or packages may be used.

[STEPS]
SECTION: MS-DIAL V 4.9.221218 starting
1. Thermo RAW files are organized in two folders - one for all positive mode raw files and another for all negative mode raw files. These files will be loaded into MS-DIAL in two separate searches. Download MSP library files for Positive and Negative mode and save them in a folder in the computer you are using to perform the searches.

SECTION: MS-DIAL V 4.9.221218 starting
2. Open MS-DIAL 
SECTION: MS-DIAL V 4.9.221218 starting
3. Click File -> New project
SECTION: MS-DIAL V 4.9.221218 starting
4. Project file path: Browse the folder with Positive or Negative raw files
Ionization type: Soft ionization (LC-MSMS)
Separation type: Chromatography (LC)
MS method type: Conventional LC/MS or data dependent MS/MS
Data type: MS1 Profile, MS/MS Centroid
Ion mode: Positive or Negative
Target omics: Metabolomics
Click "Next"
SECTION: MS-DIAL V 4.9.221218 starting
5. Analysis file path: browse to the folder with raw files, select all the apply for the search. Should include at least 3 blanks and 3 QC runs.
Choose the file type for each sample (Blank, QC, sample)
Class ID - add short group names for each replicate without adding numbers among replicates.
SECTION: Analysis parameter setting - keep default if not mentioned in this section
6. Data collection tab
Mass accuracy: MS1 tolerance 0.01 Da, MS2 tolerance 0.025 Da
 - Advanced:
Data collection parameters: retention time begin 0 min, retention time end 15 min, MS1 range begin 50 Da, MS1 range end 1000 Da, MSMS range begin 50 Da, MSMS range end 1000 Da, Isotope recognition: max charge number 2.
SECTION: Analysis parameter setting - keep default if not mentioned in this section
7. Peak Detection tab
Peak detection parameters: minimum peak height 10000 amplitude, mass slice 0.1 Da
- Advanced:
Smoothing method: linear weighted moving average
Smoothing level: 3 scan
Minimum peak width: 5 scan
SECTION: Analysis parameter setting - keep default if not mentioned in this section
8. MS2Dec tab
Deconvolution parameters: sigma window value 0.5, MS2 abundance cut off 10 amplitude.
- Advanced:
Check Exclude after precursor ion
Keep the isotope ions until 0.5 Da
Check Keep the isotopic ions w/o MS2Dec

SECTION: Analysis parameter setting - keep default if not mentioned in this section
9. Identification tab
MSP file and MS/MS identification setting: add MSP file downloaded Positive or Negative depending on the data that you loaded in the step 04. 
Retention time tolerance: 100 min, Accurate mass tolerance (MS1) 0.01  Da, Accurate mass tolerance MS/MS 0.05, Identification score cut off 80%.
- Advanced:
 add txt file of an in-house library Positive or Negative depending on the data that you loaded in the step 04. 
Retention time tolerance: 0.1 min, Accurate mass tolerance 0.01  Da, Identification score cut off 85%.
Check only report the top hit
SECTION: Analysis parameter setting - keep default if not mentioned in this section
10. Adduct tab
Check [M+H]+
Check [M+H-H2O]+
Check [2M+H]+
SECTION: Analysis parameter setting - keep default if not mentioned in this section
11. Alignment tab
Alignment parameters settings: Under result name add a name for the alignment file, reference file choose one of the QC runs, retention time tolerance 0.05 min, MS1 tolerance 0.015 Da
- Advanced:
Check remove feature based on blank information
Check keep "reference matched" metabolite features
Check keep ''suggested (w/o MS2)" metabolite features
Check keep removable features and assign the tag
Check Gap filling by compulsion
SECTION: Analysis parameter setting - keep default if not mentioned in this section
12. At the bottom
Check together with Alignment 
Click "Finish" --> processing will start.
Once the search is finished, click on the name of the file that is showing at the Alignment Navigator
Click Export - Alignment result, choose a folder to save the file, check remove features from blank, and export.
Data can be loaded into Excel/R or any other software environment for further data analysis.
SECTION: Analysis parameter setting - keep default if not mentioned in this section
13. Data Analysis: load data into Excel, filter out: m/z match FALSE and identifcations named w/o MS2. Combine postive and negative identifications removing redundancy and save as txt file. Load the combined non-redundant data into R or the online MetaboAnalyst 6.0 tool. 
SECTION: Analysis parameter setting - keep default if not mentioned in this section
14.  MetaboAnalyst 6.0 --> statistical analysis
Upload .txt data as peak intensities (check file format), no data filtering, Sample normalization: normalization by median, Data transformation: Log transformation (base 10), no Data Scaling. 
At this point there are distinct analyses that can be done, the following is provided by our core as a first-step analysis:
Chemometrics analysis: PCA, PLS-DA with importance measures using VIP.
Univariate analysis: fold change and t-test -> volcano plot (two groups comparisons).
Clustering analysis: heatmap.