Thinking... We need to answer 6 questions. We must search the Methods and Results for pathway enrichment analysis details. The article says: In "Functional annotation" and "De novo assembly and functional annotation" paragraph: "Unigene expression level was normalized by calculating reads per kb per million reads (RPKM). Comparison of unigene expression among treatments was according to DEGSeq as described by Anders and Huber and Wang et al. DEGs between treated and control samples were identified when the false discovery rate (FDR) ≤ 0.05, |log2ratio|≥1, and P-value < 0.05. The library prepared from the NT-0 pooled samples was used as calibrators to normalize the DEGs in the other six libraries. DEGs were clustered by Short Time-series Expression Miner software (STEM). The clustered profiles with a P-value ≤0.05 were considered to be significantly expressed. Then 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." Thus pathway enrichment tool: seems to be "KEGG pathway annotation using Blast all software against the KEGG database" and "Senior bubble charts were constructed using the OmicShare tools, a free online platform for data analysis". However the question "tool was used for pathway enrichment analysis" - explicitly mentioned: They used "KEGG pathway annotation using Blast all software against the KEGG database" - the tool is "Blast all software" maybe? But is "Blast all" used for enrichment? That seems to be used for KEGG annotation of DEGs. They mention "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." So the enrichment method: using KEGG database. But what tool? They say "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs". The methods section: "KEGG pathway enrichment analysis of DEGs" – They used "KEGG" database. But they don't mention a tool like DAVID or GSEA. But they mention "Short Time-series Expression Miner software (STEM)" for clustering. But that's for clustering, not for enrichment. But the enrichment step: "Then the DEGs were subjected to KEGG pathway annotation using Blast all software against the KEGG database." So they used "Blast all" (maybe "BLAST" for annotation). They also mention "Senior bubble charts were constructed using the OmicShare tools, a free online platform for data analysis". That is maybe for visualization, not for enrichment. They also mention "To understand the gene expression patterns of DEGs during fruit cracking, we performed a hierarchical cluster analysis of expression patterns with cluster software and Java TreeView." Not for enrichment. They mention "The library prepared from the NT-0 pooled samples was used as calibrators to normalize the DEGs in the other six libraries. DEGs were clustered by Short Time-series Expression Miner software (STEM). The clustered profiles with a P-value ≤0.05 were considered to be significantly expressed." So they used STEM to cluster. But the enrichment analysis is: "Then 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." Thus for enrichment analysis they used KEGG database; the tool is maybe "Blast all software" but that's annotation. The statistical test: they mention Q-value ≤0.05, but not the test used. Usually KEGG uses hypergeometric or Fisher. But the article does not specify. "Was multiple testing correction / FDR applied to enrichment analysis?" They mention Q-value. Q-value is often the false discovery rate. But they didn't mention method (Benjamini-Hochberg). They said "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." That suggests they used a Q-value threshold. They didn't explicitly mention FDR. However Q-value is a measure of false discovery rate. But they didn't say that they applied correction. It's ambiguous. The question: "Was multiple testing correction / FDR applied to enrichment analysis? Only answer Yes if the article explicitly mentions FDR, false discovery rate, Benjamini-Hochberg, or a named correction method for the enrichment analysis. A p-value threshold alone is not sufficient." They mention "Q-value ≤0.05." But they didn't specify that Q-value is derived from FDR correction. So we must say "Not described" (since not explicit). But perhaps they used "FDR" earlier: In DEG detection they used "false discovery rate (FDR) ≤ 0.05." But for enrichment analysis, they used Q-value ≤0.05. But no mention of FDR. So "Not described". Thus answer: Tool used for pathway enrichment analysis: "KEGG" or "Blast all software"? Wait "KEGG pathway annotation using Blast all software against the KEGG database." But "Blast all" might be a tool for annotation. "Blast all software" is used for KEGG annotation. They didn't mention other enrichment tools. So tool: "BLAST all" or "Blast all software". They used "BLASTx" for annotation earlier. But "Blast all" is used for KEGG annotation. The question: "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)." "Blast all software" is used for annotation. That is for enrichment. So list "Blast all". They also used "KEGG" database. They also used "OmicShare tools" to construct senior bubble charts; but that's for visualization. The question: "What tool was used for pathway enrichment analysis?" So we can answer "Blast all". But they used "STEM" for clustering of DEGs but not for enrichment. "OmicShare" maybe used to construct bubble charts. But not for enrichment. But they also used "OmicShare tools" to construct bubble charts of enrichment? That might be considered a tool for enrichment? Not exactly; it's a platform for data analysis. But they didn't explicitly state it was used for enrichment. They used "Senior bubble charts were constructed using the OmicShare tools, a free online platform for data analysis." That is a visualization tool for enrichment results. But it's not used for enrichment analysis itself. Thus for "Tool used for pathway enrichment analysis" we list "Blast all". Version of tool: Not described. Gene set libraries: "KEGG" is used. They also used "GO" and "COG" for functional annotation. But enrichment analysis was specifically "KEGG pathways" - "KEGG database." They also might have used GO for enrichment? They didn't mention performing GO enrichment, but they did "GO annotation" earlier. But for pathway enrichment, they only used KEGG. But also in the Results they mention "GO" perhaps. But not for enrichment. So gene set libraries: "KEGG". Custom or specific background gene list: Not described. They likely used all unigenes as background. Statistical tests: They didn't mention. They just said "KEGG pathways with a Q-value ≤0.05 are significantly enriched in DEGs." They didn't specify test used. But typical KEGG enrichment uses hypergeometric test or Fisher's exact test. But not mentioned. So "Not described". Multiple testing correction: They mention Q-value ≤0.05. They didn't mention FDR. So "Not described". Let's craft table with 6 columns: 1. What tool was used for pathway enrichment analysis? 2. Were enrichment tool version(s) described? 3. Which gene set libraries were used? 4. Custom background? 5. Statistical test(s) used? 6. Multiple testing correction? Table rows: We have one tool: "Blast all". But maybe also "STEM" but it's for clustering not enrichment. So only one. Now compile: Tool: Blast all Version: Not described Gene set libraries: KEGG Custom background: Not described Statistical tests: Not described Multiple testing correction: Not described But check if any other enrichment tool used: They used "STEM" for clustering. But that's not enrichment. They used "OmicShare tools" to construct bubble charts. Not for enrichment. They also used "OmicShare tools" for senior bubble charts, but not for enrichment. Thus answer. But maybe they used "OmicShare" for bubble charts. But bubble charts are for visualization. Thus final table. Let's produce table. Also we may mention that they used "KEGG" annotation and "KEGG pathways with a Q-value ≤0.05" So "KEGG" library. Now produce answer. ...done thinking. | Question | Answer | |---|---| | **What tool was used for pathway enrichment analysis?** | Blast all (used for KEGG pathway annotation of DEGs) | | **Were enrichment tool version(s) described?** | Not described | | **Which gene set libraries were used?** | KEGG | | **Does the article describe using a custom or specific background gene list for enrichment analysis?** | Not described | | **What statistical test(s) were used in enrichment analysis?** | Not described | | **Was multiple testing correction / FDR applied to enrichment analysis?** | Not described |