Thinking... We need to extract explicitly reported details about how pathway enrichment analysis was conducted. Must focus on Methods section; may refer to Results, Figures, Supplementary Materials if necessary, but only explicitly reported. We have 6 questions: 1. What tool was used for pathway enrichment analysis? List all tools or software packages used (e.g., DAVID, GSEA, Enrichr, etc.) that are explicitly mentioned in the article for enrichment analysis. Do not include tools used only for preprocessing (e.g., Salmon). So tools used for enrichment: "DAVID (Database for Annotation, Visualization and Integrated Discovery)" and "WebGestalt 2017 (WEB-based GEne SeT AnaLysis Toolkit)". Both are used. 2. Were enrichment tool version(s) described?: We saw "DAVID version 6.8" is mentioned. WebGestalt 2017; the tool maybe version 2017. The Methods mention "DAVID software version 6.8" explicitly. For WebGestalt, the Methods mention "WebGestalt toolkit version 2017" or "WebGestalt Toolkit" but not the version number? It says "WebGestalt 2017 (WEB-based GEne SeT AnaLysis Toolkit)". That's the version name. So we list: DAVID v6.8; WebGestalt 2017. If we have to list version numbers: DAVID 6.8; WebGestalt 2017. It might not have a numeric version, but 2017 indicates a release year. We'll treat it as version. 3. Which gene set libraries were used? Identify all gene set libraries or databases the authors queried. From Methods: "An Overrepresentation Enrichment Analysis (ORA) was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) version 6.8; and, WebGestalt. DAVID software revealed all Gene Ontology (GO) terms (BP, CC, and MF) and KEGG pathways of the co-expressed genes with a False Discovery Rate (FDR) of 5%. WebGestalt was used to find additional relevant KEGG pathways (FDR 5%) that may not appear in DAVID analysis." So gene set libraries used: Gene Ontology terms (BP, CC, MF). KEGG pathways. That's all. Not mention Reactome or others. But maybe other libraries used for enrichment? They might have used "Bos taurus genome was used as background". That is background. But gene set libraries: GO, KEGG. The article didn't mention other libraries. Thus gene set libraries used: GO, KEGG. That's all. 4. Does the article describe using a custom or specific background gene list for enrichment analysis? Options Yes, No, Not described. In Methods: "The Bos taurus genome was used as background for both analyses." So that indicates they used a background gene list. Is it custom or specific? They used the Bos taurus genome as background. So yes, they used a background gene list (i.e., the genome). But the question: "Does the article describe using a custom or specific background gene list for enrichment analysis?" Options Yes, No or Not described. They used the Bos taurus genome as background. That's a specific background. So answer: Yes. 5. What statistical test(s) were used in enrichment analysis? State the name(s) of the statistical test(s) used for enrichment analysis (e.g., hypergeometric test, Fisher's exact test). Only include tests linked to enrichment. In Methods: Overrepresentation Enrichment Analysis (ORA) was performed by DAVID and WebGestalt. Both likely use hypergeometric test or Fisher's exact test. The article didn't specify which test. The Methods section: "An Overrepresentation Enrichment Analysis (ORA) was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) version 6.8; and, WebGestalt 2017 (WEB-based GEne SeT AnaLysis Toolkit). DAVID software revealed all Gene Ontology terms (BP, CC, and MF) and KEGG pathways of the co-expressed genes with a False Discovery Rate (FDR) of 5%." So they didn't explicitly mention the statistical test. So answer: Not described. Wait but the question: "What statistical test(s) were used in enrichment analysis?" The article didn't mention the test. So answer: Not described. 6. Was multiple testing correction / FDR applied to enrichment analysis? The article explicitly says: "with a False Discovery Rate (FDR) of 5%" for DAVID and WebGestalt. So yes, they applied FDR. So answer: Yes. We should produce output in a table format. Let's create a table with columns: Question, Answer. But the tasks: "Please answer the following 6 questions. If more than one tool, method, or database is used, list all of them separately." So we should provide a table with rows: - Tool was used for pathway enrichment analysis: list all tools or software packages used explicitly for enrichment analysis. That would be "DAVID", "WebGestalt". - Were enrichment tool version(s) described? Provide the version numbers associated with each enrichment tool, if reported. - Which gene set libraries were used? Provide all gene set libraries or databases the authors queried. - Does the article describe using a custom or specific background gene list for enrichment analysis? Options: Yes, No, Not described. - What statistical test(s) were used in enrichment analysis? Provide the names of tests used. - Was multiple testing correction / FDR applied to enrichment analysis? Yes/No. So the table would have six rows. Each row: "Tool(s)", "Version(s)", "Gene set libraries", "Background list used", "Statistical tests", "Multiple testing correction". But the question expects each row to have answer values. Eg: | Question | Answer(s) | |---|---| | What tool was used for pathway enrichment analysis? | DAVID; WebGestalt | | Were enrichment tool version(s) described? | DAVID 6.8; WebGestalt 2017 | | Which gene set libraries were used? | Gene Ontology (BP, CC, MF); KEGG | | Does the article describe using a custom or specific background gene list for enrichment analysis? | Yes | | What statistical test(s) were used in enrichment analysis? | Not described | | Was multiple testing correction / FDR applied to enrichment analysis? | Yes | Yes. But ensure to list all tools or software packages used explicitly for enrichment analysis. They used DAVID and WebGestalt. They didn't use GSEA, Enrichr, etc. So answer accordingly. Now, double-check the article for any other mention: In the methods, "An Overrepresentation Enrichment Analysis (ORA) was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) version 6.8 [[184]70]; and, WebGestalt 2017 (WEB-based GEne SeT AnaLysis Toolkit) [[185]71]." Good. Then "DAVID software revealed all Gene Ontology (GO) terms (BP, CC, and MF) and KEGG pathways of the co-expressed genes with a False Discovery Rate (FDR) of 5%." That indicates they used FDR 5% threshold for enrichment. That is multiple testing correction. They also mention WebGestalt: "WebGestalt was used to find additional relevant KEGG pathways (FDR 5%) that may not appear in DAVID analysis." So FDR used. They didn't mention any other gene set libraries beyond GO and KEGG. They didn't mention Reactome, etc. So that's correct. They used the Bos taurus genome as background. "The Bos taurus genome was used as background for both analyses." So that indicates a specific background list. So answer Yes. Statistical test(s) used: Overrepresentation Enrichment Analysis; but test not specified. The article didn't mention hypergeometric, Fisher's, etc. So "Not described". Thus final table as requested. But let's confirm any other details: They used "TPM" for counts normalization. But that is preprocessing. So not needed. Check the Methods: "Functional enrichment analysis An Overrepresentation Enrichment Analysis (ORA) was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) version 6.8; and, WebGestalt 2017 (WEB-based GEne SeT AnaLysis Toolkit)". So they used ORA. Did they mention the statistical test? No. So answer: Not described. Thus produce final table. Be careful with the table formatting. Should be plain text with a table maybe in markdown. Let's create markdown table: | Question | Answer(s) | |---|---| | What tool was used for pathway enrichment analysis? | DAVID; WebGestalt | | Were enrichment tool version(s) described? | DAVID 6.8; WebGestalt 2017 | | Which gene set libraries were used? | Gene Ontology (BP, CC, MF); KEGG | | Does the article describe using a custom or specific background gene list for enrichment analysis? | Yes | | What statistical test(s) were used in enrichment analysis? | Not described | | Was multiple testing correction / FDR applied to enrichment analysis? | Yes | Check: For "Which gene set libraries were used?" It's GO and KEGG. That's all. Did they include GO terms (BP, CC, MF). So yes. Make sure to mention gene set libraries used: "Gene Ontology (BP, CC, MF)" and "KEGG pathways". That is all. They didn't mention other libraries. Now, confirm the article's mention of "not described" for missing details. For the test used, they didn't mention hypergeometric, etc. So answer "Not described". Thus produce final answer. ...done thinking. | Question | Answer(s) | |----------|-----------| | **What tool was used for pathway enrichment analysis?** | DAVID; WebGestalt | | **Were enrichment tool version(s) described?** | DAVID 6.8; WebGestalt 2017 | | **Which gene set libraries were used?** | Gene Ontology (BP, CC, MF); KEGG | | **Does the article describe using a custom or specific background gene list for enrichment analysis?** | Yes | | **What statistical test(s) were used in enrichment analysis?** | Not described | | **Was multiple testing correction / FDR applied to enrichment analysis?** | Yes |