Okay, let me tackle this. The user wants me to figure out how the pathway enrichment analysis was done in the provided article. They have six specific questions about the methods used. Let me go through the text again to make sure I don't miss anything. First, I need to look at the Methods section and any other relevant parts. The user mentioned that the main info is in Methods, but other sections might have clues too. Let me start by scanning the Methods again. Looking at the "Statistics" section under Methods, it says: "Descriptive statistics were calculated in R (version 3.0.2). The ratio of variant sites per nucleotide of aligned reads was calculated as follows: binomial probability distributions were performed and hypergeometric tests were calculated; each functional subset was compared to the corresponding group within the set of all coding genes (n = 14,626). This analysis allowed us to identify the gene sets that were enriched compared to similar categories among all coding genes." Hmm, so they used hypergeometric tests for the enrichment analysis. But hypergeometric test is a statistical test often used in enrichment. The background gene list is mentioned as the set of all coding genes (14,626). But the question is about pathway enrichment, like GO terms or KEGG. Wait, the "functional groups" they mention are their own categories like apoptosis, chemosensory response, etc., which they defined in the Methods under "Supporting information" part. Let me check that part again. In the Methods, under the part where they describe the gene annotations, they mention assigning genes to functional categories like 'apoptosis', 'chemosensory response', etc. These are custom categories created by the authors rather than standard pathway databases like GO or KEGG. The functional groups are listed as: apoptosis (APOP), chemosensory response (CSR), cytoskeletal/structural (CYT/STR), diverse (DIV), mitochondrial (MIT), lipid processing (LIPID), redox (ReDox), proteolysis (PROT), replication/DNA repair/transcription/translation (RRTT), signal transduction (SigT), metabolism (MET), unknown (UNK), and transport (TRP). They combined mitochondrial and redox for GSEA. So the pathway enrichment here is using their own defined functional categories, not a standard library like GO. The statistical test was hypergeometric, as stated. The background is all coding genes (n=14,626). The paper mentions using Benjamini-Hochberg FDR correction earlier in the Methods for gene-wise averages (FDR alpha=0.01), but the question is about the pathway enrichment part. The Statistics section says they used hypergeometric tests and mentions FDR? Let me check again. Wait, in the Statistics section, they mention hypergeometric tests but don't explicitly say FDR was used here. The Benjamini-Hochberg was mentioned in the part about gene-wise averages with a cutoff of 4.0. The pathway enrichment part (the functional subsets) might have used a different correction. The text doesn't specify FDR for the enrichment tests. It just says hypergeometric tests were performed. So maybe no FDR was mentioned for the pathway part. The tool: The analysis was done in R, but the specific package or tool for the hypergeometric test isn't named. The hypergeometric test in R is done via phyper or hypergeom functions, but the user might be asking for the tool/package. Since it's not specified beyond using R, the answer might be that the tool isn't named, just R version 3.0.2. The version of R is given, but the package isn't mentioned. So to answer the questions: 1. What tool was used? They used R, but the specific package isn't mentioned. The text says "calculated in R (version 3.0.2)" for descriptive stats, and hypergeometric tests were performed but without naming a tool beyond R. 2. Tool version: R 3.0.2 is mentioned, but that's the R version, not the tool's. Since the tool isn't specified (like a package), maybe the version isn't provided for the actual enrichment tool. 3. Gene set library: They used their own defined categories, not a standard like GO or KEGG. The functional groups are custom. 4. Background gene list: Yes, all coding genes (14,626). 5. Statistical test: Hypergeometric test. 6. FDR correction: The question is whether FDR was used here. The Benjamini-Hochberg was used for gene-wise averages, but the pathway enrichment part (functional subsets) doesn't mention FDR. The text says they used hypergeometric tests and compared to the background, but doesn't specify correction for multiple testing in that context. So maybe no, unless implied. The user should check, but the answer might be no. Wait, the question is about pathway enrichment analysis specifically. The Statistics section says they used hypergeometric tests. The Benjamini-Hochberg was for the gene-wise averages, not the pathway analysis. So for the pathway part, FDR might not have been applied. The user's question is whether FDR correction was used in the pathway analysis. The answer is probably no, as it's not mentioned there. Putting it all together: 1. Tool: R (but no specific package mentioned for enrichment) 2. Tool version: Not provided (R version is given but not the package's) 3. Gene set library: Custom functional categories defined by authors 4. Background: Yes, all coding genes (14,626) 5. Test: Hypergeometric test 6. FDR: No, not described for pathway analysis 1. **What tool was used for pathway enrichment analysis?** The pathway enrichment analysis was conducted using **R (version 3.0.2)**, but the specific package or tool for the hypergeometric test was not explicitly named. 2. **Was a tool version number provided?** The version of R (3.0.2) was provided, but no version number was specified for the tool (e.g., package) used to perform the enrichment analysis. 3. **What gene set library was queried?** The authors used **custom functional categories** defined by themselves, such as apoptosis (APOP), chemosensory response (CSR), mitochondrial (MIT), and others, rather than standard libraries like GO, KEGG, or Reactome. 4. **Was a background gene list defined?** Yes, the background gene list included **all coding genes (n = 14,626)**. 5. **What statistical test was used?** The statistical test was a **hypergeometric test**, as described in the "Statistics" section. 6. **Was false discovery rate (FDR) correction used?** The Benjamini-Hochberg FDR correction (alpha = 0.01) was mentioned for gene-wise averages, but **no FDR correction was explicitly described for the pathway enrichment analysis** itself. The hypergeometric test results for functional subsets did not mention FDR adjustment. | Question | Answer | |----------|--------| | 1. Tool | R (version 3.0.2, no specific package named) | | 2. Tool version | Not provided (only R version stated) | | 3. Gene set library | Custom functional categories (e.g., apoptosis, mitochondrial) | | 4. Background gene list | Yes (all coding genes, n = 14,626) | | 5. Statistical test | Hypergeometric test | | 6. FDR correction | No (not described for pathway analysis) |