This README describes the workflow for analyzing soil samples using the protocol provided in the paper.
The following diagram represents the workflow used for Lipidomics analysis in ClimGrass Experiment. Click on any box to view more detailed information about that process.
graph TD
A[Freeze dried soil samples 3x per treatment] --> C[Total Lipid Extraction Batch]
B[Class specific heavy isotope standards 13 classes, 10ng/compound] --> C
D[NIST SRM 1950 Plasma 50ul] --> C
C --> E[Sample Worklist spike External Standards, pool QC Samples]
E --> F[LC Orbitrap MS/MS]
F --> G[Untargeted analysis]
G --> H[Identification]
G --> I[Quantification]
subgraph QC [Quality Control]
style QC fill:#e6f3ff,stroke:#4da6ff,stroke-width:2px
J[Quality Control]
J --> L1[LipidQC]
click L1 "#lipidqc"
J --> L2[POOL QC]
click L2 "#pool-qc"
J --> L3[External Standard Calibration]
click L3 "#external-standard-calibration"
J --> L4[Lipid Recovery using Internal Standard]
click L4 "#lipid-recovery-using-internal-standard"
end
subgraph ID [Identification Pathway]
style ID fill:#e6ffe6,stroke:#66cc66,stroke-width:2px
H --> M1[Library search LipidSearch 5]
click M1 "https://github.com/Samratacad/Soil_Lipidomics/blob/main/methods/Dataprocessing/lipidsearch5/lipidsearch5-identification.pdf"
H --> M2[In Silico MS/MS prediction SIRIUS 5]
click M2 "https://github.com/Samratacad/Soil_Lipidomics/blob/main/methods/Dataprocessing/sirius5/SIRIUS5predictionSettings.pdf"
H --> M3[Online Search GNPS platform]
click M3 "#online-search-gnps-platform"
H --> M4[FBMN GNPS platform]
click M4 "#fbmn-gnps-platform"
end
subgraph QUANT [Quantification Pathway]
style QUANT fill:#fff0e6,stroke:#ffaa80,stroke-width:2px
I --> K1[Compound Classification using Class specific standards]
click K1 "#compound-classification-using-class-specific-standards"
I --> K2[Compound Quantification using IS Model]
click K2 "#compound-quantification-using-is-model"
end
- Sample Preparation
- Lipid Extraction
- Chromatographic & mass spectrometry Analysis
- Data Processing
- Quality Control
- Compound Identification and Quantification
Here you can find the codes, generated figures, and datafile used for figures in the respective folder
- Fig1.Lipid annotation using Library search and Prediction based approach
- Fig2.PCA and Interaction plot
- Fig3.FDR Hiararchical clustering Heatmap
- Fig4.Lipid Specificity & Shannon entropy
- Fig5.Class specific lipid concentration across treatment
- Standards
- POOL Quality Control
- S4.Quality control POOL QC
- S5.[QC figure Classwise]
- S6.Retention time CV
- External QC NIST SRM
- Lipid Recovery
- S7.[Recovery percentage internal standards]
- Hierarchical clustering of samples detailed
- Freeze-dried soil samples (3x per treatment)
- Class-specific heavy isotope standards (13 classes, 10ng/compound)
- NIST SRM 1950 Plasma (50ul) as a reference
- LC Orbitrap MS/MS
- Untargeted analysis
- Raw data files are deposited in MASSIVE database
Multiple QC steps are implemented throughout the workflow:
- Lipidomics Minimal Reporting Checklist
- NIST SRM Inter Laboratory Comparison
- POOL QC
- Lipid Recovery using Internal Standard
raw files are processed using Lipidsearch 5 and Mzmime3
- Mzmime3 files
- Lipidsearch5
- Sirius5
- Skyline transition list is prepared to perform a targeted analysis for quantification
- GNPS analysis perform online. Here are the links for files and results
Here you can find the combined annotation from all approach
- Library-search
- Prediction-based
- Compound Classification using Class-specific standards
- Compound Quantification using Ionization Efficiency Model
- LC-MS/MS: Orbitrap
- Software: LipidSearch 5, SIRIUS 5, GNPS platform
- This workflow integrates both wet-lab techniques and computational analysis.
- Quality control measures are implemented at multiple stages to ensure data reliability.
- The process allows for both targeted and untargeted analysis of lipids in soil samples.
For more detailed information on each step, please refer to the specific folder readmd file.