Thinking... We need to read the Methods, and maybe Results, to find explicitly reported details about pathway enrichment analysis. The relevant section "Pathway enrichment analysis" in Methods: Let's locate. In the provided article: In "Pathway enrichment analysis" section in Methods: Under the "Methods" heading after "Pathway enrichment analysis" we have: > Pathway enrichment analysis was performed to explore possible pathways involved in RFI phenotypic variation prior to the feedlot. Mummichog software identified the enrichment of retinol metabolic pathway (P < 0.05; Table [90]2), as being associated with FE in positive mode with 2 pathway metabolites annotated in the data. The putative compounds hit included retinoate ([91]C00777) and, either the isobaric compounds (molecular weight 284.4357): all-trans-Retinal ([92]C00376) or 11-cis-Retinal ([93]C02110) (Table [94]3). But earlier, we need to check the Methods: under "Pathway enrichment analysis" (maybe in Methods). Let's find the Methods subsection for pathway enrichment analysis. Scrolling to "Pathway enrichment analysis" within Methods. In the Methods section: we have: > Pathway enrichment analysis > Pathway enrichment analysis was performed to explore possible pathways involved in RFI phenotypic variation prior to the feedlot. Mummichog software identified the enrichment of retinol metabolic pathway (P < 0.05; Table [90]2), as being associated with FE in positive mode with 2 pathway metabolites annotated in the data. The putative compounds hit included retinoate ([91]C00777) and, either the isobaric compounds (molecular weight 284.4357): all-trans-Retinal ([92]C00376) or 11-cis-Retinal ([93]C02110) (Table [94]3). Thus we see the tool: Mummichog software. But check earlier in the methods: in "Pathway enrichment analysis" maybe they mention Mummichog as part of the Methods, but we need to confirm. Scrolling to earlier "Methods" for "Pathway enrichment analysis". Search for "Mummichog". I see under "Pathway enrichment analysis" within Methods: > Pathway enrichment analysis was performed to explore possible pathways involved in RFI phenotypic variation prior to the feedlot. Mummichog software identified the enrichment of retinol metabolic pathway (P < 0.05; Table [90]2), as being associated with FE in positive mode with 2 pathway metabolites annotated in the data. The putative compounds hit included retinoate ([91]C00777) and, either the isobaric compounds (molecular weight 284.4357): all-trans-Retinal ([92]C00376) or 11-cis-Retinal ([93]C02110) (Table [94]3). But also we saw earlier in the Methods: "Pathway enrichment analysis" is also referenced. Let's search for "Pathway enrichment analysis" in the Methods. I find: > Pathway enrichment analysis was performed to explore possible pathways involved in RFI phenotypic variation prior to the feedlot. Mummichog software identified the enrichment of retinol metabolic pathway (P < 0.05; Table [90]2), as being associated with FE in positive mode with 2 pathway metabolites annotated in the data. The putative compounds hit included retinoate ([91]C00777) and, either the isobaric compounds (molecular weight 284.4357): all-trans-Retinal ([92]C00376) or 11-cis-Retinal ([93]C02110) (Table [94]3). But the Methods section might have a separate "Pathway enrichment analysis" subsection. Let's locate the Methods subheading "Pathway enrichment analysis". In the Methods text, we see: "Pathway enrichment analysis" under the Methods heading: The article includes a "Pathway enrichment analysis" subsection within the Methods. Let's read: "Pathway enrichment analysis was performed to explore possible pathways involved in RFI phenotypic variation prior to the feedlot. Mummichog software identified the enrichment of retinol metabolic pathway (P < 0.05; Table [90]2), as being associated with FE in positive mode with 2 pathway metabolites annotated in the data. The putative compounds hit included retinoate ([91]C00777) and, either the isobaric compounds (molecular weight 284.4357): all-trans-Retinal ([92]C00376) or 11-cis-Retinal ([93]C02110) (Table [94]3)." But the "Mummichog" is mentioned there. We also see earlier in the Methods: "Pathway enrichment analysis" might be part of the "Univariate differential analysis" or "Statistical methods" but we saw. Also, the "Pathway enrichment analysis" might refer to the same Mummichog analysis, but the question is to extract details explicitly reported. Thus for each of the 6 questions: 1. Tool used for pathway enrichment analysis: The only tool explicitly mentioned for enrichment analysis is "Mummichog" software. However, we must check if there is any other tool used for pathway enrichment analysis. The article might also use the WGCNA to identify modules and annotate them with Mummichog. Mummichog is used for both annotation and enrichment. Also, maybe the article used "Pathway analysis" through "MetaboAnalyst" or something else? Let’s search for "MetaboAnalyst" in the article. Not present. So only Mummichog is mentioned. Thus the answer for "What tool was used for pathway enrichment analysis?" is "Mummichog". 2. Did the article describe the version(s) of enrichment tool? Under "Pathway enrichment analysis" the article might mention a version number for Mummichog. Let's check. The Methods text: "Mummichog software identified the enrichment of retinol metabolic pathway (P < 0.05; Table [90]2)". No version number is given. There is no mention of a version number. So answer: "Not described". 3. Which gene set libraries were used? In the pathway enrichment analysis, Mummichog uses KEGG database for metabolite annotation. The article states that Mummichog predicted retinol metabolic pathway, and they mention KEGG compounds. They refer to "KEGG" as the database for compound annotations. The article's results mention "retinol metabolic pathway" presumably from KEGG. They don't mention other gene set libraries. So we only have KEGG. They didn't mention GO, Reactome, etc. So answer: "KEGG". But let's double-check the article. In the results: "Retinol metabolism" is a KEGG pathway. So gene set library: KEGG. They didn't mention any others. They might also mention "Gene sets" like "Steroid hormone biosynthesis" (also KEGG). So yes, KEGG. Thus we list "KEGG". 4. Does the article describe using a custom or specific background gene list for enrichment analysis? In Mummichog, the background is the entire set of metabolite annotation. The article didn't mention a background list. So answer "Not described" or "No"? The question: "Does the article describe using a custom or specific background gene list for enrichment analysis?" The options: Yes, No, Not described. If not described, we write "Not described". In the Methods, I don't see any mention of background gene list. They didn't say they used a specific background or custom list. So answer: "Not described". But we should confirm if the article indicates a background gene list for enrichment analysis. Under "Pathway enrichment analysis" they used Mummichog, which uses the KEGG database for annotation. Mummichog does not typically allow specifying a background list, as it uses all features in the data as the background. They didn't mention customizing background. So "Not described". 5. What statistical test(s) were used in enrichment analysis? The article mentions that Mummichog software identified enrichment using a Fisher's exact test (they present Fisher's P value). In Table 2, they show "Fisher's P value". So the test used: Fisher's exact test. They might also use hypergeometric test. But from Mummichog documentation, it uses hypergeometric test or Fisher's exact test. The article explicitly shows "Fisher's P value" in Table 2. So the test used: Fisher's exact test. Thus we list "Fisher's exact test". 6. Was multiple testing correction / FDR applied to enrichment analysis? The article's Table 2 indicates "Fisher's P value" but no mention of FDR or multiple testing correction. They only mention P-value threshold, but no FDR. So answer: "No". The article doesn't mention FDR, Benjamini-Hochberg, etc. So "No". Thus compile a table: Columns: Tool, Version(s), Gene set library, Custom background list? (Yes/No/Not described), Statistical test, Multiple testing correction. Rows: Mummichog (only one). Provide each tool. So table: Tool | Version | Gene set library | Custom background list? | Statistical test | Multiple testing correction? Mummichog | Not described | KEGG | Not described | Fisher's exact test | No Double-check if any other enrichment tools: The article also might mention "MetaboAnalyst" or "Pathview" or "Pathway Tools". But no. Only Mummichog. Now we need to produce the output in a table format. The table has 6 columns for the 6 questions. Provide values accordingly. Let's double-check the article again to ensure no other enrichment tools were used: In the Methods, we saw "Mummichog software identified the enrichment of retinol metabolic pathway". No mention of other tools. In Results, Table 2 also shows "Retinol metabolism" (KEGG). So gene set library: KEGG. There's no mention of other libraries. So we list only KEGG. Also, version not described. Hence the final table. ...done thinking. | Question | Answer | |---|---| | What tool was used for pathway enrichment analysis? | Mummichog | | 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? | Fisher's exact test | | Was multiple testing correction / FDR applied to enrichment analysis? | No |