UHPLC-MS/MS Untargeted Metabolomics Profiling of Cecal Content in Three Chicken Breeds

This document provides a comprehensive guide to the dataset (Version 3) supporting the findings of our manuscript (ID: 1931418). To ensure clarity and traceability, the data are organized as follows. Data Integrity, Recovery, and Correction Statement The 2023 commercial metabolomics report was generated from the complete original raw data and forms the basis of the manuscript analysis. In 2026, the original .zip archives storing the raw data were found to be corrupted and could not be extracted normally. The recovered raw files are deposited in this repository for archival transparency. A limited number of recovered files have reduced scan counts due to this archive-level corruption; however, this does not affect the 2023 commercial report or the manuscript conclusions, because the 2023 report was generated from the complete original raw data. The Data_Recovery_Report.pdf documents the recovery process and confirms that all recovered files are fully consistent with the “Raw Spectral Data File” column in the ISA-Tab files. All corrections described in this repository are strictly limited to textual descriptions and do not alter any raw data, statistical results, figures, or scientific conclusions. 1. General Data Source and Folder Structure Note Folders are described below in numerical order (0–8). 0_ISA_Tab_Metadata contains the ISA-Tab metadata for the MetaboLights submission (MTBLS15811), which has been updated accordingly to correct the data processing software and statistical thresholds. 1_Raw_Data_Recovery_Records contains the raw data recovered in 2026 and related recovery records (Data_Recovery_Report.pdf). These files are deposited for archival transparency. The original .zip archives storing the raw data were corrupted and could not be extracted normally. As a result, a limited number of recovered files have reduced scan counts. However, this archive-level corruption does not affect the 2023 commercial report or the manuscript conclusions, because the 2023 commercial report was generated from the complete original raw data, and the reduced scan counts are limited to a subset of the 2026 recovered files. All recovered files are fully consistent with the “Raw Spectral Data File” column in the ISA-Tab files and the Data_Recovery_Report.pdf. 2_Commercial_Original_Plots, 3_MSMS_Identification_Spectra (MSMS = MS/MS), 4_BPC_and_QC, 5_Multivariate_Statistics, and 6_List_of_Differential_Metabolites contain results from the 2023 commercial metabolomics report, which was generated from the complete original raw data. These results form the basis of the manuscript analysis. 7_2023_Commercial_Report contains the original condensed commercial summary report (report.pdf, in Chinese) and its English summary (English_Summary_of_2023_Report.pdf). 8_Correction_Statement contains the formal correction and supporting documents submitted alongside our appeal to Frontiers, including 01_Correction_of_Methods.pdf, 02_Data_Recovery_Report.pdf, 03_English_Summary_of_2023_Report.pdf, and 04_Evidence_Timeline_and_Decisions.pdf. 02_Data_Recovery_Report.pdf is the same file as the one archived in 1_Raw_Data_Recovery_Records, and 03_English_Summary_of_2023_Report.pdf is the same file as English_Summary_of_2023_Report.pdf in Folder 7. 2. Nomenclature and Mapping Guide To ensure clarity between the commercial report and the final manuscript: Breed Name Mapping: WCJ (in commercial folders) = WCC (Wenchang Chicken, in the manuscript); BEJ (in commercial folders) = WEYC (White-ear Yellow Chicken, for future studies); YXB (in commercial folders) = RWFC (Recessive White Feather chicken, for future studies). (Note: The commercial report uses Chinese pinyin abbreviations, while the manuscript uses standardized English abbreviations). Sample Group Definitions: WCJC/BEJC/YXBC refer to the control group; WCJT/BEJT/YXBT refer to the infected group. For the WCC group, T denotes infected/treatment, corresponding to “WCC-I” in the manuscript. Time Point Mapping: WCJT1105_vs_WCJC1105 corresponds to 2 days post infection (dpi); WCJT1107_vs_WCJC1107 corresponds to 4 dpi; WCJT1110_vs_WCJC1110 corresponds to 7 dpi. 3. Important Note on Scope All statistical analyses, differential metabolite lists, and final conclusions presented in this manuscript are based exclusively on the WCC (Wenchang Chicken) group. The other two breeds (YXB and BEJ) were part of the original larger commercial project and are included in this repository primarily as raw Excel data tables, with associated transparency outputs, within the Other_Breeds_Transparency folders, to ensure complete project-level transparency and support future independent studies. No analyzed figures or conclusions pertaining to these two breeds appear in this manuscript. 4. Important Note on Figure Preparation To meet journal standards, the final figures were prepared as follows: Final Figures (PCA, OPLS-DA, OPLS-DA Permutation Tests, KEGG Enrichment Bar Plot): Generated from the original commercial data. The visual layout (fonts, colors, legend labels, grid, and ellipse fill) was standardized in Adobe Illustrator 28.0 to align with the manuscript nomenclature (e.g., WCJC1105 → WCC-C 2 dpi). The underlying data points and statistical values are identical to the original commercial report. The original, unmodified commercial plots are provided in 2_Commercial_Original_Plots for direct comparison. Regenerated Figures (Volcano Plots and Pie Charts): Re-plotted independently by the authors using the underlying Excel data tables and MS/MS differential analysis results. File Format Note: The final print-ready figures were submitted to the journal in TIFF format. For ease of preview, the same final figures are provided in this repository in PDF format. Clarification on sample naming: We would like to clarify that the sample group names used in our manuscript (“WCC-C 2 dpi” and “WCC-I 2 dpi”) correspond to the sample identifiers “WCJC1105” and “WCJT1105” used in the original commercial data files. This is solely a difference in sample annotation, not a discrepancy in the underlying data. 5. Detailed Folder-by-Folder Guide 0_ISA_Tab_Metadata Contains the structured ISA-Tab metadata files prepared for the MetaboLights submission. These serve as the global experimental guide and are fully synchronized with the MetaboLights record (MTBLS15811). The folder contains 6 standard ISA-Tab files: i_Investigation.txt: The core investigation file containing the study description, protocols, and the updated methodological corrections (MSConvert/XCMS and the two-tiered screening strategy). s_MTBLS15811.txt: Sample grouping, breed (Gallus gallus), treatment (infected vs. non-infected), and time points (2, 4, and 7 dpi). a_MTBLS15811_LC-MS_positive_reverse-phase.txt and a_MTBLS15811_LC-MS_negative_reverse-phase.txt: Instrument and chromatography parameters for positive-ion and negative-ion modes. The Raw Spectral Data File column points to the recovered raw files under 1_Raw_Data_Recovery_Records/POS/ and 1_Raw_Data_Recovery_Records/NEG/. m_MTBLS15811_LC-MS_pos_v2_maf.tsv and m_MTBLS15811_LC-MS_neg_v2_maf.tsv: Comprehensive lists of identified metabolites in the positive and negative ion modes, respectively. Note on Abundance: Due to the format of the commercial report, individual sample abundances are not embedded in these ISA-Tab files. Instead, the complete raw and normalized quantitative peak matrices (with individual sample abundances) are provided in the 5_Multivariate_Statistics/ folder. Each of these Excel files contains a Sample_Mapping worksheet that maps sample IDs to breed, treatment, time point, and original commercial code. The same Sample_Mapping.xlsx is also provided in 3_MSMS_Identification_Spectra/02_Overall_Identification_Summary/ for global reference. Clarification on Database and Search Engine Nomenclature: The manuscript's method section mentions HMDB, KEGG, and mzCloud as reference databases. However, the commercial report utilized search engines and spectral libraries (MetaDNA, BioDeepDB, MoNA, GNPS, and mzCloud) to query these underlying databases. MoNA and GNPS are public resources rather than proprietary tools. In the ISA-Tab metadata: (1) The database column is uniformly listed as KEGG because the database_identifier column contains standard KEGG compound/drug IDs (e.g., C15767, C05841); (2) The search_engine column documents the exact search engines and spectral libraries used (MetaDNA, BioDeepDB, MoNA, GNPS, mzCloud). 1_Raw_Data_Recovery_Records Contains the complete recovered raw UPLC-MS/MS data in native Thermo .raw format. POS/: 98 positive-ion raw files, covering 90 biological samples (30 per breed: WCC, RWFC, WEYC) and 8 pooled QC samples. NEG/: 98 negative-ion raw files, covering 90 biological samples (30 per breed: WCC, RWFC, WEYC) and 8 pooled QC samples. In addition to the raw data, this folder contains the formal Data_Recovery_Report.pdf, which documents the professional recovery process of the 196 raw files (30.54 GB total) from the originally corrupted .zip archives. These corrupted archives were originally generated by the commercial metabolomics service provider (Nuomi Metabolomics) in 2023. With assistance from the original commercial service provider and Shanghai Yinguan Biomedical Technology Co., Ltd.—a registered biotechnology technical service provider—the complete dataset was successfully recovered. All recovered files are fully consistent with the “Raw Spectral Data File” column in a_MTBLS15811_LC-MS_positive_reverse-phase.txt and a_MTBLS15811_LC-MS_negative_reverse-phase.txt. Important Note on Split Archives: The original 196 raw files total approximately 30.54 GB (uncompressed). Due to their large size, the 1_Raw_Data_Recovery_Records folder has been split into multiple volumes using 7-Zip (named 1_Raw_Data_Recovery_Records.zip.001, .002, .003..., totaling approximately 24.4 GB). Downloaders must download all these volumes into the same folder and open the .001 file with 7-Zip or WinRAR to extract the complete raw data. Do not attempt to extract a single volume separately, as it will fail and result in incomplete data. 2_Commercial_Original_Plots Contains the original, unmodified commercial plots corresponding to the manuscript figures. These plots are provided for direct comparison with the final, standardized figures submitted to the journal. The folder is divided into three subfolders: 01_NEG/: Negative-ion-specific plots. Each comparison group is organized in its own subfolder (e.g., WCJT1105_vs_WCJC1105/, WCJT1107_vs_WCJC1107/, WCJT1110_vs_WCJC1110/; see the Nomenclature and Mapping Guide in the Data Description for time-point mapping). Each subfolder contains: Commercial_WCC_PCA_Score_Plot.pdf, Commercial_WCC_OPLS_DA_Score_Plot.pdf, Commercial_WCC_OPLS_DA_Permutation_Test.pdf. 02_POS/: Positive-ion-specific plots. The structure is identical to 01_NEG/, with the same comparison groups and plot types. 03_Combined_MS2/: Combined positive- and negative-ion MS2-level plots. Each time point is organized in its own subfolder (e.g., WCJT1105_vs_WCJC1105/ for 2 dpi, etc.). Each subfolder contains: Commercial_WCC_Volcano_Plot.pdf, Commercial_WCC_KEGG_Enrichment_Bar_Plot.pdf. All plots in this folder are the original, unmodified versions provided by the commercial service. The final manuscript figures were standardized in Adobe Illustrator 28.0 for nomenclature and visual consistency, but the underlying data and statistical values remain identical to these original plots. 3_MSMS_Identification_Spectra This folder contains the comprehensive MS/MS identification library for the entire project. 01_Raw_MSMS_Spectra/: Contains all 1,149 individual MS/MS spectra compiled into a single PDF (All_MSMS_Spectra.pdf). This serves as the complete commercial identification library. Although these raw spectral plots are not presented in the manuscript, they provide the foundational evidence for all metabolite identifications. 02_Overall_Identification_Summary/: Contains the overall identification matrix (Overall_Identified_Metabolite_Matrix.xlsx) and the identified metabolite list (Identified_Metabolite_List.xlsx) in Excel format. Crucially, a separate Sample_Mapping.xlsx file is provided in this folder to clearly map each sample ID to its original code, breed, treatment, time point, and manuscript label. This is the same Sample_Mapping.xlsx file provided in 5_Multivariate_Statistics/. The WCC-specific identifications derived from this library form the basis of the differential metabolite analysis in the manuscript. (Note: The manuscript’s conclusions, differential metabolite lists, and pathway enrichments are based exclusively on the WCC group. The inclusion of the other two breeds is solely for maintaining the integrity of the original complete dataset for transparency and future independent studies.) 4_BPC_and_QC Contains base peak chromatograms (BPCs), QC validation plots, and total ion chromatogram (TIC) data. The folder is divided into two subfolders: NEG/ (negative-ion mode) and POS/ (positive-ion mode). The structure of POS/ is identical to NEG/. Each ion mode folder contains the following four subfolders: 01_WCC_BPC_Plots/: Contains the individual BPC plots specifically for the WCC group (WCC_Individual_BPC.pdf), demonstrating the chromatographic quality of the samples used in the manuscript. 02_Overall_Batch_QC_Validation/: Contains QA_QC_PCA_Score_Plot.pdf, QC_Sample_PCA_Score_Plot.pdf, and RSD_Distribution.pdf. This folder demonstrates the analytical reproducibility of the entire experimental batch (all three breeds). These pooled QC samples were prepared by mixing equal aliquots of all samples, and their tight clustering and low RSD values prove the high quality of the chromatographic data, including the WCC samples used in the manuscript. (Note: The commercial report labeled these “QA”, referring to the same pooled QC samples.) 03_WCC_TIC_Plots/: Contains the total ion chromatograms (TICs) specifically for the WCC group. 04_Other_Breeds_Transparency/: Contains BPC and TIC data for the broader project, provided solely for transparency. It includes two subfolders: BPC_Other_Breeds_Transparency/ (containing 00_Overall_Merged_BPC_All_Breeds/, 01_BEJ_Individual_BPC/, and 02_YXB_Individual_BPC/) and TIC_Other_Breeds_Transparency/. 5_Multivariate_Statistics Contains multivariate models and quantitative peak matrices. Divided into NEG/ (negative-ion mode) and POS/ (positive-ion mode), with identical structures. 01_WCC_Analysis/: Final manuscript-ready figures and underlying data for the WCC group. Each time point (2, 4, and 7 dpi) includes 01_PCA/ (score plot and model table) and 02_OPLS_DA/ (score plot, permutation test, and model table). Although the 2023 commercial report performed exploratory PLS-DA, the final manuscript deliberately selected PCA and OPLS-DA. No PLS-DA figures or tables are included in the manuscript. This is fully consistent with the data deposited in the Zenodo repository. 02_WCC_Quantitative_Peak_Matrices/: Contains the complete quantitative peak tables specifically extracted for the WCC group: WCC_metabolome_neg.xlsx (and WCC_metabolome_pos.xlsx under POS/). Each file contains both raw and normalized worksheets, with columns including the WCC samples and the global pooled QC samples (QC1-QC8). A Sample_Mapping worksheet is included to clarify sample identities. These files serve as the direct input for all statistical models and enable full independent reproduction of the manuscript’s results. 03_Other_Breeds_Transparency/: Data from the broader commercial project provided for full transparency. It includes: 00_Overall_Batch_Analysis_All_Breeds/, 01_Quantitative_Peak_Matrices/, and 02_Multivariate_Model_Results/. 6_List_of_Differential_Metabolites Contains the differential metabolite analysis results, divided into 01_NEG/, 02_POS/, and 03_MS2_Differential_Analysis_Combined/. (Note: KEGG enrichment results have been integrated into 03_MS2_Differential_Analysis_Combined/ to avoid data duplication and are not presented as a separate top-level folder.) 01_NEG/ and 02_POS/: MS1-level differential analysis for negative- and positive-ion modes, provided as background or supplementary data. Each contains: 001_WCC_MS1_Differential_Analysis/ and 002_Other_Breeds_Transparency/. 03_MS2_Differential_Analysis_Combined/ (Manuscript Core): Contains the definitive MS2-level differential analysis and KEGG enrichment results, which are the exact data presented in the manuscript. 001_WCC_Analysis/: Core data supporting the manuscript’s conclusions. Each time point (WCJT1105_vs_WCJC1105/, WCJT1107_vs_WCJC1107/, WCJT1110_vs_WCJC1110/) includes: 001_Differential_Analysis/ (quantification lists, filtered differential metabolites, full analysis results, statistical results, final volcano plots, and metabolite pie charts) and 002_KEGG_Enrichment/ (final KEGG enrichment bar plots and underlying data tables). The differential metabolite screening strictly followed the two-tiered strategy: P < 0.05 and VIP > 1.0 for 2 dpi; VIP > 1.0, |log2 fold change| > 1, and FDR-adjusted P < 0.05 for 4 dpi and 7 dpi. For KEGG enrichment, no pathway reached statistical significance at P < 0.05; pathways with P < 0.2 were considered as showing a trend towards enrichment. 002_Other_Breeds_Transparency/: Original commercial MS2 analysis and KEGG enrichment outputs for the other two breeds. It includes: 00_Overall_Summary/, 01_BEJ_Analysis/, and 02_YXB_Analysis/. 7_2023_Commercial_Report Folder 7_2023_Commercial_Report contains the original commercial report (report.pdf, in Chinese) generated by the commercial metabolomics service provider (Nuomi Metabolomics) in 2023 based on the complete dataset. Please note that this report.pdf is a highly condensed summary report, not the complete detailed dataset. The complete detailed datasets (including all raw spectra, quantification matrices, and detailed analysis results for all three chicken breeds) are extracted and reorganized into folders 0 to 6 in this repository. An English summary (English_Summary_of_2023_Report.pdf) is also provided in this folder to ensure full comprehension for the editorial review. 8_Correction_Statement Contains the formal correction and supporting documents submitted alongside our appeal to Frontiers. This folder includes: 01_Correction_of_Methods.pdf (detailing four unintentional textual corrections regarding the data processing software, the two-tiered differential screening strategy, the exclusion of PLS-DA, and the KEGG pathway analysis). 02_Data_Recovery_Report.pdf (verifying the successful recovery of the 196 raw files, 30.54 GB. This is the same file as the one archived in 1_Raw_Data_Recovery_Records.) 03_English_Summary_of_2023_Report.pdf (an English summary of the original 2023 commercial report. This is the same file as English_Summary_of_2023_Report.pdf in Folder 7.) 04_Evidence_Timeline_and_Decisions.pdf (chronological evidence from initial acceptance to final rejection). These corrections are strictly limited to textual descriptions and do not alter any raw data, statistical results, figures, or the scientific conclusions of the manuscript. All statements above are supported by the deposited files and the Data_Recovery_Report.pdf.