issue while running pathway_errorbar()

[DaliBAmor]

I applied your script on my data. As you can see in the metadata I have a column called traitment with ( CTRL , TR1 ,TR2 and TR4) ko_abundance_file <- "pred_metagenome_unstrat.tsv" kegg_abundance <- ko2kegg_abundance(ko_abundance_file) # Or use data(kegg_abundance) metadata <- read_delim("metadata.txt", delim = "\t", escape_double = FALSE, trim_ws = TRUE)

when I use edgeR : daa_results_df <- pathway_daa(abundance = kegg_abundance, metadata = metadata, group = "traitment", daa_method = "edgeR", select = NULL, p.adjust = "BH", reference = NULL)

I have this error: Sample names extracted. Identifying matching columns in metadata... Matching columns identified: sample . This is important for ensuring data consistency. Using all columns in abundance. Converting abundance to a matrix... Reordering metadata... Converting metadata to a matrix and data frame... Extracting group information... Processing data with edgeR method... Initializing edgeR object... Calculating normalization factors... Estimating common dispersions... Disp = 0.04671 , BCV = 0.2161 edgeR processing completed. Error in pathway_daa(abundance = kegg_abundance, metadata = metadata, : Notice: There are no statistical significances detected. This is not an error, but it might indicate that your data does not contain any values passing the set significance threshold (p<=0.05). You may refer to the tutorial's FAQ for further help and suggestions.

when I change the method : ALDEx2

daa_results_df <- pathway_daa(abundance = kegg_abundance, metadata = metadata, group = "traitment", daa_method = "ALDEx2", select = NULL, p.adjust = "BH", reference = NULL) daa_annotated_results_df <- pathway_annotation(pathway = "KO", daa_results_df = daa_results_df, ko_to_kegg = TRUE) p <- pathway_errorbar(abundance = kegg_abundance, daa_results_df = daa_annotated_results_df, Group = metadata$traitment, ko_to_kegg = TRUE, p_values_threshold = 0.05, order = "pathway_class", select = NULL, p_value_bar = TRUE, colors = NULL, x_lab = "pathway_name") I have this error : The following pathways are missing annotations and have been excluded: ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00740, ko04940, ko00010, ko00195, ko03450, ko00760, ko00920, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko04974, ko04976, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, ko00061, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, ko05322, ko04964, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, ko00940, ko00941, ko01053, ko00100, ko00945, ko01057, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko00312, ko05020, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00400, ko00350, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko00965, ko03008, ko00290, ko04614, ko03410, ko05010, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00020, ko00710, ko02060, ko00340, ko00785, ko00550, ko00650, ko02030, ko03320, ko04612, ko04621, ko04622, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00232, ko00230, ko04122, ko00540, ko05014, ko03020, ko03010, ko00140, ko05100, ko00860, ko05410, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, ko00901, ko00903, ko00471, ko00472, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko04640, ko03420, ko00770, ko02040, ko00670, ko04920, ko04144, ko00930, ko04112, ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00740, ko04940, ko00010, ko00195, ko03450, ko00760, ko00920, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko04974, ko04976, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, ko00061, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, ko05322, ko04964, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, ko00940, ko00941, ko01053, ko00943, ko00100, ko00945, ko01057, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko00312, ko05020, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00400, ko00350, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko00965, ko03008, ko00290, ko04614, ko03410, ko05010, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00020, ko00710, ko02060, ko00340, ko00785, ko00550, ko00650, ko02030, ko03320, ko04612, ko04621, ko04622, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00232, ko00230, ko04122, ko00540, ko05014, ko00660, ko03020, ko00982, ko03010, ko00140, ko05100, ko00860, ko05410, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, ko00901, ko00903, ko00471, ko00472, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko04640, ko03420, ko00770, ko02040, ko00670, ko04920, ko04144, ko00930, ko04112 You can use the 'pathway_annotation' function to add annotations for these pathways. There are more than two 'group' columns in the 'daa_results_df' data frame. As a result, it is not possible to compute the log2 fold values. The 'p_value_bar' has been automatically set to FALSE. The 'method' column in the 'daa_results_df' data frame contains more than one method. Please filter it to contain only one method. The 'group1' or 'group2' column in the 'daa_results_df' data frame contains more than one group. Please filter each to contain only one group. Error in pathway_errorbar(abundance = kegg_abundance, daa_results_df = daa_annotated_results_df, : Visualization with 'pathway_errorbar' cannot be performed because there are no features with statistical significance. For possible solutions, please check the FAQ section of the tutorial.

when I use : DESeq2 daa_results_df <- pathway_daa(abundance = kegg_abundance, metadata = metadata, group = "traitment", daa_method = "DESeq2", select = NULL, p.adjust = "BH", reference = NULL)

I have this error : Starting pathway annotation... DAA results data frame is not null. Proceeding... KO to KEGG is set to TRUE. Proceeding with KEGG pathway annotations... We are connecting to the KEGG database to get the latest results, please wait patiently.

The number of statistically significant pathways exceeds the database's query limit. Please consider breaking down the analysis into smaller queries or selecting a subset of pathways for further investigation.

Returning DAA results filtered annotation data frame...

and then when I run this command ; p <- pathway_errorbar(abundance = kegg_abundance, daa_results_df = daa_annotated_results_df, Group = metadata$traitment, ko_to_kegg = TRUE, p_values_threshold = 0.05, order = "pathway_class", select = NULL, p_value_bar = TRUE, colors = NULL, x_lab = "pathway_name") I have this error : The following pathways are missing annotations and have been excluded: ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko04940, ko00010, ko00195, ko03450, ko00760, ko00920, ko00310, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko04974, ko04976, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, ko00061, ko03070, ko00253, ko03060, ko00500, ko05322, ko04964, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00270, NA, ko00940, ko00941, ko01053, NA, ko00100, ko00945, ko01057, NA, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko03050, ko00040, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko05020, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, NA, ko00052, ko00053, ko00051, ko00400, ko00350, ko00480, ko00643, ko00642, ko00720, ko00960, ko02020, ko00120, ko00965, ko00290, ko04614, NA, ko03410, ko05010, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00020, ko00710, ko00196, ko02060, ko00785, ko00550, ko00650, ko02030, ko03320, ko04612, ko04622, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00232, ko00230, ko04122, ko00540, ko05014, ko00660, ko03020, NA, ko00982, ko03010, ko05100, ko00860, ko05410, ko00330, ko00908, ko03430, ko00906, ko00900, ko00901, ko00903, ko00472, ko00473, ko05111, NA, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko04640, ko03420, ko00770, ko02040, ko00670, ko04144, ko00930, ko04112, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00740, ko04940, ko00195, ko03450, ko00760, ko00920, ko00311, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko05110, ko04974, ko04976, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, NA, ko00061, ko03070, ko03060, ko00380, ko00500, ko05120, ko05322, ko04964, ko00625, ko00624, ko00626, ko00621, ko00622, ko00940, ko00941, ko01053, ko00943, ko00945, ko01057, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko00281, ko03013, ko04626, ko05200, ko00312, ko05020, ko05143, ko00520, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00400, ko00350, ko00480, ko00643, ko00640, ko00960, ko02020, ko00120, ko00965, ko03008, ko00290, ko04614, ko05010, ko05012, ko00980, ko00983, ko05150, ko00791, ko00020, ko00710, ko00196, ko02060, ko00340, ko00785, ko00650, ko02030, ko03320, ko04612, ko04621, ko04622, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00232, ko00230, ko04122, ko00540, ko05014, ko00660, ko00982, ko03010, ko00140, ko05100, ko00860, ko05410, ko00330, ko00908, ko03430, ko00906, ko00900, ko00901, ko00903, ko00471, ko00472, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko04640, ko03420, ko00770, ko02040, ko00670, ko04920, ko04144, ko04112, ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, NA, ko00740, ko04940, ko00010, ko00195, ko03450, ko00920, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko04974, ko04976, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, NA, ko00061, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, NA, ko04964, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, ko00940, ko00941, ko01053, NA, ko00100, ko00945, ko01057, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko00312, ko05020, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, ko00053, ko00051, ko00401, ko00400, ko00350, ko00480, ko00643, ko00642, ko00640, ko00960, ko02020, ko00120, ko00965, ko03008, ko00290, ko04614, ko05010, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00020, ko00196, ko02060, ko00340, ko00785, ko00550, ko00650, ko02030, ko03320, ko04612, ko04621, ko04622, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00232, ko00230, ko04122, ko00540, ko05014, ko00660, ko00982, ko03010, ko00140, ko05100, ko00860, ko05410, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, ko00901, NA, ko00903, ko00471, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko04640, ko03420, ko00770, ko02040, ko00670, ko04920, ko04144, ko00930, ko04112, ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, NA, ko00740, ko00010, ko00195, ko00760, ko00311, ko00310, ko04146, ko04141, ko04142, ko05142, ko04973, ko00510, ko04974, ko00450, ko01051, ko00524, ko00300, ko00260, ko03440, ko00750, ko00950, ko00592, ko00590, NA, ko00061, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, NA, ko00625, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, NA, ko01053, ko00100, ko05016, NA, ko00071, ko00072, ko05219, ko05215, ko01055, ko04910, ko00531, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko00121, ko04914, ko00430, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko03013, ko05200, ko00312, ko05020, ko05143, ko00523, ko00520, ko00521, ko05145, ko00052, ko00051, ko00401, ko00400, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko00290, NA, ko03410, ko05010, ko00980, ko00410, ko00983, ko05150, ko00790, ko05131, ko00020, ko00710, ko00196, ko02060, ko00340, ko00785, ko00550, ko02030, ko03320, ko04612, ko04621, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00230, ko04122, ko00540, ko05014, ko00660, ko03020, NA, ko00982, ko03010, ko00140, ko05100, ko00860, ko00908, ko03430, ko00906, ko00900, NA, ko00903, ko00471, ko00473, ko05111, NA, NA, ko04210, ko00240, ko04011, ko00944, ko03420, ko02040, ko00670, ko04920, ko00930, ko04112, ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00740, ko04940, ko00010, ko00195, ko03450, ko00760, ko00920, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko04974, ko04976, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, ko00061, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, ko05322, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, ko00940, ko00941, ko01053, ko00943, ko00100, ko00945, ko01057, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, NA, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko00281, ko00280, ko03013, ko04626, ko00312, ko05020, ko05143, ko00523, ko00521, ko05146, ko05145, ko05144, ko00052, ko00053, ko00051, ko00401, ko00400, ko00350, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko00965, ko03008, ko00290, ko04614, ko03410, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00020, ko00710, ko02060, ko00340, ko00785, ko00550, ko00650, ko02030, ko03320, ko04612, ko04621, ko04622, ko00910, ko00830, ko00780, ko00511, ko00970, ko00030, ko00232, ko00230, ko04122, ko00540, ko05014, ko00660, ko03020, ko00982, ko03010, ko00140, ko05100, ko00860, ko05410, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, ko00901, ko00903, ko00471, ko00472, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko04640, ko03420, ko00770, ko02040, ko00670, ko04920, ko04144, ko00930, ko04112, ko05340, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00740, ko04940, ko00010, ko00195, ko03450, ko00760, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko04260, ko05142, ko04973, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, ko00061, ko03070, ko00253, ko03060, ko05120, ko04964, ko00625, ko00624, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, ko00940, ko00941, ko01053, NA, ko00100, ko00945, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, NA, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko05200, ko00312, ko05020, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00400, ko00350, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko03008, ko03410, ko05010, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00710, ko02060, ko00340, ko00550, ko00650, ko02030, ko03320, ko04612, ko04621, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00230, ko00540, ko05014, ko00660, ko03020, ko00982, ko03010, ko00140, ko05100, ko00860, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, ko00903, ko00471, ko00472, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko03420, ko00770, ko02040, ko00670, ko04920, ko00930, ko04112, ko05340, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00010, ko00195, ko03450, ko00920, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, ko00061, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, ko04964, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, NA, ko00940, ko00941, ko01053, ko00100, ko00945, ko05016, NA, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, NA, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko00312, ko05020, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00350, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko03008, ko00290, NA, ko03410, ko05010, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00710, ko02060, ko00340, ko00785, ko00550, ko02030, ko03320, ko04612, ko04621, ko04622, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00230, ko04122, ko00540, ko05014, ko00660, ko03020, NA, ko00982, ko03010, ko00140, ko05100, ko00860, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, ko00903, ko00471, ko00472, ko00473, ko05111, NA, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko03420, ko00770, ko02040, ko00670, ko04920, ko00930, ko04112, ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00740, ko04940, ko00010, ko00195, ko03450, ko00760, ko00920, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko04974, ko04976, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko03440, ko00950, ko00592, ko00591, ko00590, NA, ko00061, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, ko05322, ko04964, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, ko00940, ko00941, ko01053, ko00100, ko00945, ko01057, ko05016, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, NA, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko00312, ko05020, ko05143, ko00523, ko00521, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00400, ko00350, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00965, ko03008, ko00290, ko04614, ko03410, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00020, ko00710, ko02060, ko00340, ko00785, ko00550, ko00650, ko02030, ko04612, ko04621, ko04622, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00232, ko00230, ko04122, ko00540, ko05014, ko00660, ko03020, ko00982, ko03010, ko00140, ko05100, ko00860, ko05410, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, ko00901, ko00903, ko00471, ko00472, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko03420, ko00770, ko02040, ko00670, ko04920, ko04144, ko00930, ko04112, ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00440, ko00250, ko00740, ko04940, ko00010, ko03450, ko00760, ko00920, ko00310, ko04146, ko00600, ko04142, ko00604, ko04260, ko05142, ko00510, ko04974, ko04976, ko00450, ko01051, ko00565, ko00300, ko05222, ko05416, ko00260, ko00190, ko00750, ko00950, ko00592, ko00591, ko00590, NA, ko00061, ko03070, ko00253, ko03060, ko00500, ko05120, ko05322, ko04964, ko00625, ko00624, ko00626, ko00620, ko00623, ko00622, ko00270, ko00940, ko00941, ko01053, ko00100, ko00945, ko01057, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko04910, ko00531, NA, ko00360, ko00633, ko00630, ko00363, ko00364, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00400, ko00350, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko00965, ko00290, ko04614, ko03410, ko05010, ko05012, ko00980, ko00410, ko05150, ko00791, ko00790, ko00020, ko00710, ko02060, ko00340, ko00785, ko00550, ko00650, ko02030, ko03320, ko04612, ko04621, ko04622, ko00910, ko00460, ko00830, ko00780, ko00511, ko00970, ko00030, ko00232, ko00230, ko04122, ko00540, ko05014, ko00660, ko03020, ko00982, ko03010, ko00140, ko00860, ko05410, ko00330, ko03430, ko00906, ko00900, ko00901, ko00903, ko00471, ko00472, ko05111, ko04210, ko04011, ko04113, ko04070, ko04640, ko03420, ko00770, ko02040, ko00670, ko04920, ko04144, ko00930, ko04112, ko05340, ko00564, ko00680, ko00562, ko03030, ko00561, ko00250, NA, ko00740, ko04940, ko00010, ko00195, ko03450, ko00920, ko00311, ko00310, ko04146, ko00600, ko04141, ko04142, ko00604, ko04260, ko05142, ko04973, ko00510, ko00450, ko01051, ko00565, ko00524, ko00300, ko05222, ko05416, ko00260, ko00190, ko03440, ko00750, ko00950, ko00592, ko00591, ko00590, NA, ko03070, ko00253, ko03060, ko00380, ko00500, ko05120, NA, ko04964, ko00625, ko00624, ko00627, ko00626, ko00621, ko00620, ko00623, ko00622, ko00270, ko00940, ko01053, ko00100, ko00945, ko05016, ko00071, ko00072, ko05219, ko05215, ko05211, ko05210, ko01055, ko04910, ko00531, NA, ko00360, ko00633, ko00630, ko00363, ko00364, ko05130, ko04115, ko00121, ko04914, ko00430, ko02010, ko00130, ko03050, ko00361, ko00040, ko00730, ko00362, ko01040, ko00603, ko03018, ko00281, ko00280, ko03013, ko04626, ko05200, ko00312, ko05020, ko05143, ko00523, ko00520, ko00521, ko05146, ko05145, ko00052, ko00053, ko00051, ko00401, ko00400, NA, ko00350, ko00480, ko00643, ko00642, ko00640, ko00720, ko00960, ko02020, ko00120, ko03008, ko00290, ko04614, ko03410, ko05010, ko05012, ko00980, ko00410, ko00983, ko05150, ko00791, ko00790, ko05131, ko00020, ko00710, ko02060, ko00340, ko00785, ko00550, ko00650, ko02030, ko03320, ko04612, ko04621, ko00910, ko00460, ko00830, ko00511, ko00970, ko00030, NA, ko00230, ko04122, ko00540, ko00660, ko03020, ko00982, ko00140, ko05100, ko00860, ko00330, ko00908, ko04930, ko03430, ko00906, ko00900, NA, ko00903, ko00471, ko00473, ko05111, ko04210, ko00240, ko04011, ko00944, ko04113, ko04070, ko03420, ko00770, ko02040, ko00670, ko00930, ko04112 You can use the 'pathway_annotation' function to add annotations for these pathways. The 'method' column in the 'daa_results_df' data frame contains more than one method. Please filter it to contain only one method. The 'group1' or 'group2' column in the 'daa_results_df' data frame contains more than one group. Please filter each to contain only one group. Error in pathway_errorbar(abundance = kegg_abundance, daa_results_df = daa_annotated_results_df, : Visualization with 'pathway_errorbar' cannot be performed because there are no features with statistical significance. For possible solutions, please check the FAQ section of the tutorial.

when I use lesfer

daa_results_df <- pathway_daa(abundance = kegg_abundance, metadata = metadata, group = "traitment", daa_method = "Lefser", select = NULL, p.adjust = "BH", reference = NULL) daa_annotated_results_df <- pathway_annotation(pathway = "KO", daa_results_df = daa_results_df, ko_to_kegg = TRUE)

I have this error : Starting pathway annotation... DAA results data frame is not null. Proceeding... KO to KEGG is set to TRUE. Proceeding with KEGG pathway annotations... Error in pathway_annotation(pathway = "KO", daa_results_df = daa_results_df, : No statistically significant biomarkers found. 'Statistically significant biomarkers' refer to those biomarkers that demonstrate a significant difference in expression between different groups, as determined by a statistical test (p_adjust < 0.05 in this case). You might consider re-evaluating your experiment design or trying alternative statistical analysis methods. Consult with a biostatistician or a data scientist if you are unsure about the next steps.

when I use LinDA:

daa_results_df <- pathway_daa(abundance = kegg_abundance, metadata = metadata, group = "traitment", daa_method = "LinDA", select = NULL, p.adjust = "BH", reference = "CTRL")

daa_annotated_results_df <- pathway_annotation(pathway = "KO", daa_results_df = daa_results_df, ko_to_kegg = TRUE)

i HAVE THIS: Starting pathway annotation... DAA results data frame is not null. Proceeding... KO to KEGG is set to TRUE. Proceeding with KEGG pathway annotations... We are connecting to the KEGG database to get the latest results, please wait patiently.

The number of statistically significant pathways exceeds the database's query limit. Please consider breaking down the analysis into smaller queries or selecting a subset of pathways for further investigation.

Returning DAA results filtered annotation data frame...

nothing is working for me :( the input file was generated by using picrust2 pipeline metadata file:

ko abundance file structure (pred_metagenome_unstrat.tsv) :