Okay, let's tackle this. The user wants me to examine how the pathway enrichment analysis was conducted in the provided scientific article. They have specific questions about the methods, so I need to go through the text carefully. First, the user mentioned focusing on the Methods section but also looking elsewhere if needed. Let me start by reviewing the Methods section again. The article's Methods section has several subsections: De novo assembly and functional annotation, RT-qPCR validation, Statistical analysis, etc. Looking at the De novo assembly and functional annotation part: It says they used BLASTx against Nr, UniProt, KEGG, GO, and COG databases. Then, for DEGs, they mention KEGG pathway annotation using Blast all software against KEGG. The key part here is the pathway enrichment analysis. The text states: "The DEGs were subjected to KEGG pathway annotation using Blast all software against the KEGG database. KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." So, for the first question: What tool was used? It mentions "Blast all software" and KEGG database. Wait, Blast all might be a typo or mistranslation? Maybe they meant BLAST? Or perhaps a specific tool? Alternatively, since they mention KEGG, maybe they used KEGG's own tools like KAAS (KEGG Automatic Annotation Server) or another tool. Alternatively, the text says "Blast all software against the KEGG database". Hmm, not sure. Alternatively, maybe they used a different tool like clusterProfiler in R? But the article doesn't specify. Wait, in the Methods, under De novo assembly and functional annotation, the exact line is: "The DEGs were subjected to KEGG pathway annotation using Blast all software against the KEGG database." The term "Blast all software" might be a translation issue. Maybe they used a custom script or a standard tool. Alternatively, maybe they used the KEGG Automatic Annotation Server (KAAS). Alternatively, perhaps they used a tool like DAVID or another enrichment tool. Since the article doesn't specify a tool name beyond Blast all software, maybe that's the answer. Alternatively, maybe "Blast all" is a misunderstanding, but since the user is asking for the tool, I have to go with what's written. Alternatively, maybe the tool used is part of the KEGG pathway analysis mentioned, but the exact name isn't given beyond "Blast all software". Wait, in the same section, they mention "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." The Q-value here is typically a FDR-corrected p-value. So maybe they used a hypergeometric test or Fisher's exact test, common in pathway enrichment. The tool might be part of the KEGG pathway analysis tools. Alternatively, they might have used a software like clusterProfiler in R, but that's not mentioned here. The article says "Blast all software", which is unclear. Maybe the authors used a custom approach? Alternatively, perhaps they used a tool like Blast2GO? Not sure. Since the exact tool isn't named beyond "Blast all software", maybe the answer is "Blast all software" as per the text, but I need to check again. Looking at the other questions: the gene set library is KEGG, since they're doing KEGG pathway annotation. The statistical test: the text mentions Q-value which is FDR, so likely hypergeometric test with FDR correction. The background gene list would presumably be all the genes in the study, but the article doesn't explicitly state that. Let me go through each question step by step. 1. Tool used: The text says "KEGG pathway annotation using Blast all software against the KEGG database." So the tool is referred to as "Blast all software". That might be a mistranslation or typo. Alternatively, maybe it's referring to BLAST followed by some analysis. Since it's unclear, but the article states that, I have to report "Blast all software" as the tool. Alternatively, maybe it's a tool named "BlastAll" but that's speculative. Alternatively, perhaps they used the KEGG's own tools, but the article doesn't specify beyond that line. 2. Tool version: The article doesn't mention any version numbers for the tool used. So answer is no. 3. Gene set library: Definitely KEGG, as they used the KEGG database for pathway annotation. 4. Background gene list: The article says "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." The background would likely be all the genes in the reference transcriptome, but it's not explicitly stated. The DEGs were compared against the background, but the text doesn't mention defining a specific background list. So answer is "not described". 5. Statistical test: The Q-value is mentioned, which is typically from FDR correction. The test itself for enrichment is likely hypergeometric test, which is standard for pathway enrichment. The article says "KEGG pathways with a Q-value ≤0.05 are significantly enriched", so the test is hypergeometric, but the exact test isn't named. Since they mention Q-value, perhaps a hypergeometric test with FDR correction. But the question is about the statistical test used before FDR. The answer would be hypergeometric test, but the article doesn't explicitly name it. Alternatively, they might have used Fisher's exact test. Since it's not specified beyond "Q-value", maybe the answer is not described, but the presence of Q-value suggests it's part of the enrichment method used. Hmm, tricky. The article says "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." So the test must have been done such that Q-value is computed. The method for computing Q-value (like using FDR correction on p-values from hypergeometric) is implied. But the actual test is hypergeometric. However, the user is asking what statistical test was used for enrichment analysis. Since they mention Q-value, but not the test, maybe it's not described? Or perhaps it's part of the KEGG tool's method. Since the article doesn't explicitly state the test (e.g., hypergeometric), the answer might be "not described". 6. FDR correction: The Q-value is stated as the cutoff, which is FDR. So yes, FDR was used. Wait, let me check again. The text says "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." The Q-value here refers to the FDR-corrected p-value. So the FDR correction was applied, so answer is yes. Putting it all together: 1. Tool: The article mentions "Blast all software" for KEGG pathway annotation. That's the tool listed. 2. Version: No version provided. 3. Gene set library: KEGG. 4. Background gene list: Not described. 5. Statistical test: Not explicitly stated, but inferred as hypergeometric test. However, since the article doesn't name it, the answer should be "not described". Wait, but maybe the tool they used (Blast all software) includes the statistical test. Since the user is asking for the test used, and the article doesn't specify beyond Q-value, perhaps the answer is "not described". Alternatively, the process might be that after mapping genes to KEGG pathways, they calculated the enrichment using a hypergeometric test. Since that's standard, maybe that's assumed, but the article doesn't state it, so "not described". Hmm, the user wants to know if the statistical test was mentioned. Since the article only mentions Q-value (which is a result of FDR correction on p-values, but the underlying test isn't named), I think the answer is "not described". So final answers: 1. Blast all software 2. Not provided 3. KEGG 4. Not described 5. Not described 6. Yes (because Q-value is FDR corrected) Wait, but the question 6 is whether FDR was used. Since they mention Q-value (which is FDR), the answer is yes. Therefore: 1. Tool: Blast all software 2. No version 3. KEGG 4. Not described 5. Not described 6. Yes 1. **Tool used for pathway enrichment analysis**: Blast all software 2. **Tool version number provided**: No 3. **Gene set library queried**: KEGG 4. **Background gene list defined**: Not described 5. **Statistical test used**: Not described (implied hypergeometric test but not explicitly stated) 6. **False discovery rate (FDR) correction**: Yes (Q-value ≤0.05 indicates FDR correction) ### Summary Table: | Question | Answer | |--------------------------------------------------------------------------|-------------------------------------------------------------------------| | 1. Tool used for pathway enrichment analysis | Blast all software | | 2. Tool version number provided | Not described | | 3. Gene set library queried (e.g., GO, KEGG) | KEGG | | 4. Background gene list defined for analysis | Not described | | 5. Statistical test used (e.g., hypergeometric, Fisher’s exact) | Not described (Q-value cutoff implies a statistical test like hypergeometric, but not explicitly named) | | 6. FDR correction used to control false positives | Yes (Q-value ≤0.05 denotes FDR correction) |