Okay, let's see. The user wants me to figure out how the pathway enrichment analysis was conducted in this article. I need to go through the provided text and look for the Methods section or any other sections that mention the analysis. The user has specific questions, so I should answer each one step by step. First, the question is about the tool used for pathway enrichment. The article mentions GSEA (Gene Set Enrichment Analysis) in the figure legends, like in S4 Fig which refers to TCM and TEM-related gene sets from GSEA. The main text also talks about GSEA when discussing TCM and TEM-related genes not being enriched. So the tool is likely GSEA. Next, the version number. The article doesn't explicitly state the version of GSEA used. The methods section in the Supporting Information (S1 Fig) lists Cufflinks and Cuffdiff, but those are for RNA-seq analysis. There's no mention of GSEA version in the text provided. For the gene set library, the article refers to "TCM and TEM-related gene sets" and mentions "gene sets from previous studies". Since they used GSEA, common libraries like MSigDB are typically used. The text mentions "gene sets" but doesn't specify the exact library. However, since they mention TCM (central memory T cells) and TEM (effector memory T cells) which are part of MSigDB's hallmark or immunology collections, maybe MSigDB. But the exact library isn't named here. Wait, the user's question 3 is about the library, like GO, KEGG, etc. The article doesn't specify, so maybe it's not described? Wait, in the main text under the Results section, they mention using gene sets from previous studies. For example, "genes previously found to be highly expressed in TRM cells" so maybe they used custom gene sets? The article might have used gene sets specific to TRM cells from previous publications rather than standard libraries. But the exact library isn't mentioned. Hmm, so perhaps the gene set library isn't described explicitly here. Regarding the background gene list, usually in GSEA the background is all genes on the platform. The Methods in S1 Fig mention using Cuffdiff for differential expression, but the pathway analysis part doesn't specify the background. The user's question 4 is whether a background was defined. The article might not have specified, so that's not described. The statistical test used: GSEA typically uses the Kolmogorov-Smirnov statistic. The text mentions "enrichment plots" and "P-value <0.05 and FDR q-value <0.25". So the test is probably the standard GSEA statistical approach, but the exact test isn't named. However, since they mention P-values and FDR, maybe they used a permutation-based test for GSEA. For FDR correction: The article says "significantly enriched at P-value <0.05 and FDR q-value <0.25". So yes, FDR correction was used. Wait, looking back: in the main text under the Results, they mention "the significant overlap seen between genes involved in TRM cell retention and those in the TGF-β induced signature". The Methods in the supporting info (S1 Fig) explains the RNA-seq analysis but not the enrichment. The part about GSEA is in the figure legends, like S4 Fig mentions "FDR q-value <0.25". The main text also states "none of the gene sets were significantly enriched at P-value <0.05 and FDR q-value <0.25" for TCM/TEM. So FDR was used. Putting it all together: 1. Tool: GSEA (Gene Set Enrichment Analysis) 2. Version: Not provided 3. Gene set library: Not explicitly named, possibly custom or MSigDB but not stated 4. Background gene list: Not described 5. Statistical test: Likely Kolmogorov-Smirnov based on GSEA's method, but not specified 6. FDR correction: Yes, using q-value <0.25 Wait, but the user's question 3 is about the library (like GO, KEGG, etc). The article refers to "gene sets from previous studies" but doesn't name a specific library. So maybe the answer is that it's not described? Because the exact library isn't mentioned. The authors might have used custom gene sets based on prior literature rather than a standard database. Since they mention "gene sets" but don't name the source, I have to say it's not described. Similarly, the statistical test: GSEA uses a Kolmogorov-Smirnov-like statistic with permutations, but if the text doesn't mention it, then maybe they just say "not described" for question 5? Wait, the article says "enrichment plots for the (A) TCM and TEM-related up-regulated gene sets... the curved lines show the cumulative enrichment score." That's consistent with GSEA's enrichment plots. But the statistical test details aren't given. So perhaps the answer is that the statistical test isn't described beyond using GSEA, but since GSEA's method is known, maybe they assume it's known. However, the user is asking what was used in the article. Since the article doesn't explicitly state the statistical test (like mentioning K-S test or permutation), then the answer is not described. Wait, the main text says "enrichment analysis?" Wait, in the Results section: "the significant overlap seen between genes involved in TRM cell retention and those in the TGF-β induced signature suggests..." So perhaps the overlap was assessed via overrepresentation analysis (ORA) using a tool like DAVID? But the figure mentions GSEA. The S4 Fig specifically refers to GSEA enrichment plots, so GSEA is the tool. Therefore, the answers are: 1. GSEA 2. Not provided 3. Not described (since they might have used custom gene sets from previous studies without naming a standard library) 4. Not described 5. Not described (but implied as part of GSEA's method) 6. Yes, FDR q-value <0.25 Wait, the question 5 is about the statistical test used. Since GSEA uses a specific statistical approach (like Kolmogorov-Smirnov statistic with permutations), but the article didn't mention it. The user wants to know what test they used. Since the article only mentions "enrichment plots" and "P-value" and "FDR", maybe they used a standard test, but without explicit mention, it's not described. Alternatively, since GSEA is the tool, the test is part of GSEA's methodology. But the user wants to know what was stated in the article. Since the article doesn't specify beyond using GSEA, then the answer would be "Gene Set Enrichment Analysis (GSEA)", but the test isn't separately described beyond that. So maybe the answer is "not described" for question 5. Alternatively, perhaps in the Methods section (S1 Fig), they used Cuffdiff for differential expression, and GSEA for enrichment, but the actual GSEA method's statistical details aren't provided. So for question 5, the statistical test used for enrichment is the one inherent to GSEA, but since it's not explicitly stated in the text provided, the answer is "not described". So final answers based on the given text: 1. GSEA 2. No version mentioned 3. Not described (they used gene sets from previous studies but didn't name the library) 4. Not described 5. Not described (though GSEA's test is implicit) 6. Yes, FDR correction was used (q-value <0.25) 1. **Tool used for pathway enrichment analysis**: GSEA (Gene Set Enrichment Analysis) 2. **Tool version number**: Not provided 3. **Gene set library queried**: Not described (gene sets were mentioned as from "previous studies" but no specific library like GO or KEGG was named) 4. **Background gene list defined**: Not described 5. **Statistical test used**: Not explicitly described (implied to be part of GSEA's methodology, e.g., Kolmogorov-Smirnov-like statistic with permutations, but not stated in the text) 6. **False discovery rate (FDR) correction**: Yes (FDR q-value < 0.25 was used to control false positives) | Question | Answer | |----------|--------| | 1. Tool used | GSEA (Gene Set Enrichment Analysis) | | 2. Tool version | Not provided | | 3. Gene set library | Not described (gene sets from previous studies, but library not named) | | 4. Background gene list | Not described | | 5. Statistical test | Not described (implied to be part of GSEA's methodology) | | 6. FDR correction | Yes (q-value < 0.25) |