Examining some cruzi infected HeLa cells.

1 Introduction

Let us check out some new cruzi infections following the deletion of a specific gene.

I thought I also did the interrogation of the CLBrener transcriptome, but that appears untrue. I think I may have forgotten to copy the genome in place…

2 Human annotation information

I have a pretty new genome downloaded (202509), so I will (for now) just let my annotation function grab whatever it thinks is reasonable. It chose the 202410 set. Seems good to me.

hs_annot <- load_biomart_annotations()

## The biomart annotations file already exists, loading from it.

tc_annot <- load_gff_annotations("~/libraries/genome/gff/tcruzi_all.gff",
                                 type = "mRNA", id_col = "Parent")

## Returning a df with 24 columns and 23305 rows.

rownames(tc_annot) <- gsub(x = make.names(tc_annot[["Name"]], unique = TRUE),
                           pattern = "\\.\\d+$", replacement = "")
esmer_db <- "org.Tcruzi.CL.Brener.Esmeraldo.like.v68.eg.db"
library(esmer_db, character.only = TRUE)

## Loading required package: AnnotationDbi

## Loading required package: stats4

## Loading required package: BiocGenerics

## 
## Attaching package: 'BiocGenerics'

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## Loading required package: Biobase

## Welcome to Bioconductor
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##     select

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esmer_db <- get0(esmer_db)
all_keytypes <- keytypes(esmer_db)
wanted_idx <- grepl(x = all_keytypes, pattern = "^ANNOT_")
wanted_fields <- all_keytypes[wanted_idx]
nonesmer_db <- "org.Tcruzi.CL.Brener.Non.Esmeraldo.like.v68.eg.db"
unas_db <- "org.Tcruzi.CL.Brener.v68.eg.db"

tc_esmer <- load_orgdb_annotations(esmer_db, keytype = "gid", fields = wanted_fields)

## Unable to find CDSNAME, setting it to ANNOT_EXTERNAL_DB_NAME.

## Unable to find CDSCHROM in the db, removing it.

## Unable to find CDSSTRAND in the db, removing it.

## Unable to find CDSSTART in the db, removing it.

## Unable to find CDSEND in the db, removing it.

## Extracted all gene ids.

## Attempting to select: ANNOT_EXTERNAL_DB_NAME, GENE_TYPE, ANNOT_AA_SEQUENCE_ID, ANNOT_ANNOTATED_GO_COMPONENT, ANNOT_ANNOTATED_GO_FUNCTION, ANNOT_ANNOTATED_GO_ID_COMPONENT, ANNOT_ANNOTATED_GO_ID_FUNCTION, ANNOT_ANNOTATED_GO_ID_PROCESS, ANNOT_ANNOTATED_GO_PROCESS, ANNOT_ANTICODON, ANNOT_APOLLO_LINK_OUT, ANNOT_APOLLO_TRANSCRIPT_DESCRIPTION, ANNOT_CDS, ANNOT_CDS_LENGTH, ANNOT_CHROMOSOME, ANNOT_CODING_END, ANNOT_CODING_START, ANNOT_EC_NUMBERS, ANNOT_EC_NUMBERS_DERIVED, ANNOT_END_MAX, ANNOT_EXON_COUNT, ANNOT_EXTERNAL_DB_NAME, ANNOT_EXTERNAL_DB_VERSION, ANNOT_FIVE_PRIME_UTR_LENGTH, ANNOT_GENE_CONTEXT_END, ANNOT_GENE_CONTEXT_START, ANNOT_GENE_END_MAX, ANNOT_GENE_END_MAX_TEXT, ANNOT_GENE_ENTREZ_ID, ANNOT_GENE_ENTREZ_LINK_DISPLAYTEXT, ANNOT_GENE_ENTREZ_LINK_URL, ANNOT_GENE_EXON_COUNT, ANNOT_GENE_HTS_NONCODING_SNPS, ANNOT_GENE_HTS_NONSYN_SYN_RATIO, ANNOT_GENE_HTS_NONSYNONYMOUS_SNPS, ANNOT_GENE_HTS_STOP_CODON_SNPS, ANNOT_GENE_HTS_SYNONYMOUS_SNPS, ANNOT_GENE_LOCATION_TEXT, ANNOT_GENE_NAME, ANNOT_GENE_ORTHOLOG_NUMBER, ANNOT_GENE_ORTHOMCL_NAME, ANNOT_GENE_PARALOG_NUMBER, ANNOT_GENE_PREVIOUS_IDS, ANNOT_GENE_PRODUCT, ANNOT_GENE_START_MIN, ANNOT_GENE_START_MIN_TEXT, ANNOT_GENE_TOTAL_HTS_SNPS, ANNOT_GENE_TRANSCRIPT_COUNT, ANNOT_GENE_TYPE, ANNOT_GENOMIC_SEQUENCE_LENGTH, ANNOT_GENUS_SPECIES, ANNOT_HAS_MISSING_TRANSCRIPTS, ANNOT_INTERPRO_DESCRIPTION, ANNOT_INTERPRO_ID, ANNOT_IS_DEPRECATED, ANNOT_IS_PSEUDO, ANNOT_ISOELECTRIC_POINT, ANNOT_LOCATION_TEXT, ANNOT_MAP_LOCATION, ANNOT_MCMC_LOCATION, ANNOT_MOLECULAR_WEIGHT, ANNOT_NCBI_TAX_ID, ANNOT_ORTHOMCL_LINK, ANNOT_OVERVIEW, ANNOT_PFAM_DESCRIPTION, ANNOT_PFAM_ID, ANNOT_PIRSF_DESCRIPTION, ANNOT_PIRSF_ID, ANNOT_PREDICTED_GO_COMPONENT, ANNOT_PREDICTED_GO_FUNCTION, ANNOT_PREDICTED_GO_ID_COMPONENT, ANNOT_PREDICTED_GO_ID_FUNCTION, ANNOT_PREDICTED_GO_ID_PROCESS, ANNOT_PREDICTED_GO_PROCESS, ANNOT_PRIMARY_KEY, ANNOT_PROB_MAP, ANNOT_PROB_MCMC, ANNOT_PROSITEPROFILES_DESCRIPTION, ANNOT_PROSITEPROFILES_ID, ANNOT_PROTEIN_LENGTH, ANNOT_PROTEIN_SEQUENCE, ANNOT_PROTEIN_SOURCE_ID, ANNOT_PSEUDO_STRING, ANNOT_SEQUENCE_DATABASE_NAME, ANNOT_SEQUENCE_ID, ANNOT_SIGNALP_PEPTIDE, ANNOT_SMART_DESCRIPTION, ANNOT_SMART_ID, ANNOT_SNPOVERVIEW, ANNOT_SO_ID, ANNOT_SO_TERM_DEFINITION, ANNOT_SO_TERM_NAME, ANNOT_SO_VERSION, ANNOT_START_MIN, ANNOT_STRAND, ANNOT_STRAND_PLUS_MINUS, ANNOT_SUPERFAMILY_DESCRIPTION, ANNOT_SUPERFAMILY_ID, ANNOT_THREE_PRIME_UTR_LENGTH, ANNOT_TIGRFAM_DESCRIPTION, ANNOT_TIGRFAM_ID, ANNOT_TM_COUNT, ANNOT_TRANS_FOUND_PER_GENE_INTERNAL, ANNOT_TRANSCRIPT_INDEX_PER_GENE, ANNOT_TRANSCRIPT_LENGTH, ANNOT_TRANSCRIPT_LINK, ANNOT_TRANSCRIPT_PRODUCT, ANNOT_TRANSCRIPT_SEQUENCE, ANNOT_TRANSCRIPTS_FOUND_PER_GENE, ANNOT_UNIPROT_IDS, ANNOT_UNIPROT_LINKS

## 'select()' returned 1:1 mapping between keys and columns

tc_nonesmer <- load_orgdb_annotations(nonesmer_db, keytype = "gid", fields = wanted_fields)

## 

## Unable to find CDSNAME, setting it to ANNOT_EXTERNAL_DB_NAME.

## Unable to find CDSCHROM in the db, removing it.

## Unable to find CDSSTRAND in the db, removing it.

## Unable to find CDSSTART in the db, removing it.

## Unable to find CDSEND in the db, removing it.

## Extracted all gene ids.

## Attempting to select: ANNOT_EXTERNAL_DB_NAME, GENE_TYPE, ANNOT_AA_SEQUENCE_ID, ANNOT_ANNOTATED_GO_COMPONENT, ANNOT_ANNOTATED_GO_FUNCTION, ANNOT_ANNOTATED_GO_ID_COMPONENT, ANNOT_ANNOTATED_GO_ID_FUNCTION, ANNOT_ANNOTATED_GO_ID_PROCESS, ANNOT_ANNOTATED_GO_PROCESS, ANNOT_ANTICODON, ANNOT_APOLLO_LINK_OUT, ANNOT_APOLLO_TRANSCRIPT_DESCRIPTION, ANNOT_CDS, ANNOT_CDS_LENGTH, ANNOT_CHROMOSOME, ANNOT_CODING_END, ANNOT_CODING_START, ANNOT_EC_NUMBERS, ANNOT_EC_NUMBERS_DERIVED, ANNOT_END_MAX, ANNOT_EXON_COUNT, ANNOT_EXTERNAL_DB_NAME, ANNOT_EXTERNAL_DB_VERSION, ANNOT_FIVE_PRIME_UTR_LENGTH, ANNOT_GENE_CONTEXT_END, ANNOT_GENE_CONTEXT_START, ANNOT_GENE_END_MAX, ANNOT_GENE_END_MAX_TEXT, ANNOT_GENE_ENTREZ_ID, ANNOT_GENE_ENTREZ_LINK_DISPLAYTEXT, ANNOT_GENE_ENTREZ_LINK_URL, ANNOT_GENE_EXON_COUNT, ANNOT_GENE_HTS_NONCODING_SNPS, ANNOT_GENE_HTS_NONSYN_SYN_RATIO, ANNOT_GENE_HTS_NONSYNONYMOUS_SNPS, ANNOT_GENE_HTS_STOP_CODON_SNPS, ANNOT_GENE_HTS_SYNONYMOUS_SNPS, ANNOT_GENE_LOCATION_TEXT, ANNOT_GENE_NAME, ANNOT_GENE_ORTHOLOG_NUMBER, ANNOT_GENE_ORTHOMCL_NAME, ANNOT_GENE_PARALOG_NUMBER, ANNOT_GENE_PREVIOUS_IDS, ANNOT_GENE_PRODUCT, ANNOT_GENE_START_MIN, ANNOT_GENE_START_MIN_TEXT, ANNOT_GENE_TOTAL_HTS_SNPS, ANNOT_GENE_TRANSCRIPT_COUNT, ANNOT_GENE_TYPE, ANNOT_GENOMIC_SEQUENCE_LENGTH, ANNOT_GENUS_SPECIES, ANNOT_HAS_MISSING_TRANSCRIPTS, ANNOT_INTERPRO_DESCRIPTION, ANNOT_INTERPRO_ID, ANNOT_IS_DEPRECATED, ANNOT_IS_PSEUDO, ANNOT_ISOELECTRIC_POINT, ANNOT_LOCATION_TEXT, ANNOT_MAP_LOCATION, ANNOT_MCMC_LOCATION, ANNOT_MOLECULAR_WEIGHT, ANNOT_NCBI_TAX_ID, ANNOT_ORTHOMCL_LINK, ANNOT_OVERVIEW, ANNOT_PFAM_DESCRIPTION, ANNOT_PFAM_ID, ANNOT_PIRSF_DESCRIPTION, ANNOT_PIRSF_ID, ANNOT_PREDICTED_GO_COMPONENT, ANNOT_PREDICTED_GO_FUNCTION, ANNOT_PREDICTED_GO_ID_COMPONENT, ANNOT_PREDICTED_GO_ID_FUNCTION, ANNOT_PREDICTED_GO_ID_PROCESS, ANNOT_PREDICTED_GO_PROCESS, ANNOT_PRIMARY_KEY, ANNOT_PROB_MAP, ANNOT_PROB_MCMC, ANNOT_PROSITEPROFILES_DESCRIPTION, ANNOT_PROSITEPROFILES_ID, ANNOT_PROTEIN_LENGTH, ANNOT_PROTEIN_SEQUENCE, ANNOT_PROTEIN_SOURCE_ID, ANNOT_PSEUDO_STRING, ANNOT_SEQUENCE_DATABASE_NAME, ANNOT_SEQUENCE_ID, ANNOT_SIGNALP_PEPTIDE, ANNOT_SMART_DESCRIPTION, ANNOT_SMART_ID, ANNOT_SNPOVERVIEW, ANNOT_SO_ID, ANNOT_SO_TERM_DEFINITION, ANNOT_SO_TERM_NAME, ANNOT_SO_VERSION, ANNOT_START_MIN, ANNOT_STRAND, ANNOT_STRAND_PLUS_MINUS, ANNOT_SUPERFAMILY_DESCRIPTION, ANNOT_SUPERFAMILY_ID, ANNOT_THREE_PRIME_UTR_LENGTH, ANNOT_TIGRFAM_DESCRIPTION, ANNOT_TIGRFAM_ID, ANNOT_TM_COUNT, ANNOT_TRANS_FOUND_PER_GENE_INTERNAL, ANNOT_TRANSCRIPT_INDEX_PER_GENE, ANNOT_TRANSCRIPT_LENGTH, ANNOT_TRANSCRIPT_LINK, ANNOT_TRANSCRIPT_PRODUCT, ANNOT_TRANSCRIPT_SEQUENCE, ANNOT_TRANSCRIPTS_FOUND_PER_GENE, ANNOT_UNIPROT_IDS, ANNOT_UNIPROT_LINKS

## 'select()' returned 1:1 mapping between keys and columns

tc_unas <- load_orgdb_annotations(unass_db, keytype = "gid", fields = wanted_fields)

## Error: object 'unass_db' not found

tc_more <- rbind(tc_esmer$genes, tc_nonesmer$genes, tc_more$genes)

## Error: object 'tc_more' not found

tc_annot <- merge(tc_annot, tc_more, by = "row.names")

## Error in h(simpleError(msg, call)): error in evaluating the argument 'y' in selecting a method for function 'merge': object 'tc_more' not found

rownames(tc_annot) <- tc_annot[["gid"]]
tc_annot[["gid"]] <- NULL

2.1 Load cruzi GO data similarly

tc_esmer_go <- load_orgdb_go(esmer_db, keytype = "GID")

## This is an orgdb, good.

## 'select()' returned 1:many mapping between keys and columns
## 'select()' returned 1:many mapping between keys and columns

tc_nonesmer_go <- load_orgdb_go(nonesmer_db, keytype = "GID")

## This is an orgdb, good.
## 'select()' returned 1:many mapping between keys and columns
## 'select()' returned 1:many mapping between keys and columns

tc_unas_go <- load_orgdb_go(unas_db, keytype = "GID")

## 

## This is an orgdb, good.

## 'select()' returned 1:many mapping between keys and columns
## 'select()' returned 1:many mapping between keys and columns

tc_go <- rbind(tc_esmer_go, tc_nonesmer_go, tc_unas_go)
tc_go <- tc_go[, c("GO", "GID")]
colnames(tc_go) <- c("GO", "ID")

3 Sample sheet

I asked for one from Najib/Amalie but unless I am mistaken it has not arrived. That is not a problem, given two helpful things: April provides one, I also named the directories so that the sample IDs are built in; so I will just make a fake one for now and then merge in whatever I get from them…

sample_sheet <- "sample_sheets/all_samples.xlsx"

plot_meta_sankey(as.data.frame(extract_metadata(sample_sheet)), factors = c("background", "exp_number"))

## Did not find the condition column in the sample sheet.

## Filling it in as undefined.

## Did not find the batch column in the sample sheet.

## Filling it in as undefined.

## Checking the state of the condition column.

## Checking the state of the batch column.

## Checking the condition factor.

## Warning: attributes are not identical across measure variables; they will be dropped

## Warning: The `size` argument of `element_rect()` is deprecated as of ggplot2 3.4.0.
## ℹ Please use the `linewidth` argument instead.
## ℹ The deprecated feature was likely used in the ggsankey package.
##   Please report the issue at <https://github.com/davidsjoberg/ggsankey/issues>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated.

## A sankey plot describing the metadata of 14 samples,
## including 19 out of 0 nodes and traversing metadata factors:
## background, exp_number.

4 Adding some metadata

Let us see how well my preprocess gatherer does…

new_meta <- gather_preprocessing_metadata(sample_sheet, species = c("hg38_115", "tcruzi_all"))

## Did not find the condition column in the sample sheet.

## Filling it in as undefined.

## Did not find the batch column in the sample sheet.

## Filling it in as undefined.

## Checking the state of the condition column.

## Checking the state of the batch column.

## Checking the condition factor.

## Error in dispatch_regex_search(meta, search, replace, input_file_spec, : object 'found' not found

head(new_meta$new_meta)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'x' in selecting a method for function 'head': object 'new_meta' not found

5 The primary data structure

hs_se <- create_se(new_meta[["new_meta"]], gene_info = hs_annot[["gene_annotations"]],
                   file_column = "hisat_count_table_hg38_115") %>%
  set_conditions(fact = "background") %>%
  set_batches(fact = "exp_number")

## Error in h(simpleError(msg, call)): error in evaluating the argument 'exp' in selecting a method for function 'set_batches': error in evaluating the argument 'exp' in selecting a method for function 'set_conditions': object 'new_meta' not found

tc_se <- create_se(new_meta[["new_meta"]], gene_info = tc_annot,
                   file_column = "hisat_count_table_tcruzi_all") %>%
  set_conditions(fact = "background") %>%
  set_batches(fact = "exp_number")

## Error in h(simpleError(msg, call)): error in evaluating the argument 'exp' in selecting a method for function 'set_batches': error in evaluating the argument 'exp' in selecting a method for function 'set_conditions': object 'new_meta' not found

tc_se <- subset_se(tc_se, nonzero = 5000)

## Error: object 'tc_se' not found

plot_libsize(hs_se)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_libsize': object 'hs_se' not found

plot_nonzero(hs_se)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_nonzero': object 'hs_se' not found

plot_libsize(tc_se)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_libsize': object 'tc_se' not found

plot_nonzero(tc_se)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_nonzero': object 'tc_se' not found

hs_replicated <- subset_se(hs_se, min_replicates = 3, fact = "condition")

## Error: object 'hs_se' not found

tc_replicated <- subset_se(tc_se, min_replicates = 3, fact = "condition") %>%
  subset_se(nonzero = 10000)

## Error: object 'tc_se' not found

6 Sample clustering

6.1 Human

devtools::load_all("~/hpgltools")

## ℹ Loading hpgltools
## in method for 'plot_topn_gsea' with signature 'gse="all_cprofiler"': no definition for class "all_cprofiler"
## 
## in method for 'plot_topn_gsea' with signature 'gse="clusterprofiler_result"': no definition for class "clusterprofiler_result"

## Warning: multiple methods tables found for 'annotation'

## Warning: multiple methods tables found for 'annotation<-'

hs_norm <- normalize(hs_replicated, transform = "log2", convert = "cpm",
                        norm = "quant", filter = TRUE)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'normalize': object 'hs_replicated' not found

plot_disheat(hs_norm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'input_data' in selecting a method for function 'plot_heatmap': object 'hs_norm' not found

plot_corheat(hs_norm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'input_data' in selecting a method for function 'plot_heatmap': object 'hs_norm' not found

plot_pca(hs_norm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_pca': object 'hs_norm' not found

hs_rnorm <- normalize(hs_replicated, transform = "log2", convert = "cpm",
                      norm = "quant", filter = TRUE)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'normalize': object 'hs_replicated' not found

plot_disheat(hs_rnorm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'input_data' in selecting a method for function 'plot_heatmap': object 'hs_rnorm' not found

plot_pca(hs_rnorm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_pca': object 'hs_rnorm' not found

hs_rbnorm <- normalize(hs_replicated, transform = "log2", convert = "cpm",
                       filter = TRUE, batch = "svaseq")

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'normalize': object 'hs_replicated' not found

plot_pca(hs_rbnorm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_pca': object 'hs_rbnorm' not found

6.2 Parasite

tc_norm <- normalize(tc_replicated, transform = "log2", convert = "cpm",
                     norm = "quant", filter = TRUE)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'normalize': object 'tc_replicated' not found

plot_disheat(tc_norm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'input_data' in selecting a method for function 'plot_heatmap': object 'tc_norm' not found

plot_corheat(tc_norm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'input_data' in selecting a method for function 'plot_heatmap': object 'tc_norm' not found

plot_pca(tc_norm, plot_labels = TRUE)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_pca': object 'tc_norm' not found

tc_rnorm <- normalize(tc_se, transform = "log2", convert = "cpm",
                      norm = "quant", filter = TRUE)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'normalize': object 'tc_se' not found

plot_disheat(tc_rnorm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'input_data' in selecting a method for function 'plot_heatmap': object 'tc_rnorm' not found

plot_pca(tc_rnorm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_pca': object 'tc_rnorm' not found

tc_rbnorm <- normalize(tc_se, transform = "log2", convert = "cpm",
                       filter = TRUE, batch = "svaseq")

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'normalize': object 'tc_se' not found

plot_pca(tc_rbnorm)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'data' in selecting a method for function 'plot_pca': object 'tc_rbnorm' not found

7 DE?

I am not thinking we will see many genes of interest.

hs_keepers <- list(
  "ab_vs_control" = c("AB10", "control"),
  "ko_vs_control" = c("ko7", "control"),
  "ko_vs_wt" = c("ko7", "wt"),
  "ab_vs_wt" = c("AB10", "wt"),
  "ab_vs_ko" = c("AB10", "ko7"))
hs_de <- all_pairwise(hs_replicated, filter = TRUE, model_fstring = "~ 0 + condition",
                      model_svs = "svaseq")

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'pData': object 'hs_replicated' not found

hs_de

## Error: object 'hs_de' not found

hs_tables <- combine_de_tables(hs_de, keepers = hs_keepers, excel = "excel/hs_tables.xlsx")

## Deleting the file excel/hs_tables.xlsx before writing the tables.

## Error: object 'hs_de' not found

hs_tables

## Error: object 'hs_tables' not found

hs_sig <- extract_significant_genes(hs_tables, excel = "excel/hs_sig.xlsx")

## Error: object 'hs_tables' not found

hs_sig

## Error: object 'hs_sig' not found

conditions(tc_replicated)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'exp' in selecting a method for function 'conditions': object 'tc_replicated' not found

tc_keepers <- list(
  "ab_vs_wt" = c("AB10", "wt"),
  "ko_vs_wt" = c("ko7", "wt"),
  "ab_vs_ko" = c("AB10", "ko7"))
tc_de <- all_pairwise(tc_replicated, filter = TRUE, model_fstring = "~ 0 + condition",
                      model_svs = "svaseq")

## Error in h(simpleError(msg, call)): error in evaluating the argument 'object' in selecting a method for function 'pData': object 'tc_replicated' not found

tc_de

## Error: object 'tc_de' not found

tc_tables <- combine_de_tables(tc_de, keepers = tc_keepers, excel = "excel/tc_tables.xlsx")

## Error: object 'tc_de' not found

tc_tables

## Error: object 'tc_tables' not found

tc_sig <- extract_significant_genes(tc_tables, excel = "excel/tc_sig.xlsx")

## Error: object 'tc_tables' not found

tc_sig

## Error: object 'tc_sig' not found

8 Try some ontology searching via clusterProfiler

ko_wt_up <- tc_sig[["deseq"]][["ups"]][["ko_vs_wt"]]

## Error: object 'tc_sig' not found

ko_wt_all <- tc_tables[["data"]][["ko_vs_wt"]]

## Error: object 'tc_tables' not found

tc_esmer_up_cp <- simple_clusterprofiler(
  ko_wt_up, de_table = ko_wt_all, orgdb = esmer_db, orgdb_to = "GID",
  organism = "tcruzi")

## Error: object 'ko_wt_all' not found

tc_nonesmer_up_cp <- simple_clusterprofiler(
  ko_wt_up, de_table = ko_wt_all, orgdb = nonesmer_db, orgdb_to = "GID",
  organism = "tcruzi")

## Error: object 'ko_wt_all' not found

tc_unas_up_cp <- simple_clusterprofiler(
  ko_wt_up, de_table = ko_wt_all, orgdb = unas_db, orgdb_to = "GID",
  organism = "tcruzi")

## Error: object 'ko_wt_all' not found

length_db <- as.data.frame(rowData(tc_se))

## Error in h(simpleError(msg, call)): error in evaluating the argument 'x' in selecting a method for function 'as.data.frame': error in evaluating the argument 'x' in selecting a method for function 'rowData': object 'tc_se' not found

length_db[["gid"]] <- rownames(length_db)

## Error in h(simpleError(msg, call)): error in evaluating the argument 'x' in selecting a method for function 'rownames': object 'length_db' not found

length_db <- length_db[, c("gid", "width")]

## Error: object 'length_db' not found

tc_up_gs <- simple_goseq(ko_wt_up, go_db = tc_go, length_db = length_db, min_xref = 10)

## Error: object 'ko_wt_up' not found

mf_enr <- tc_up_gs[["mf_enrich"]]

## Error: object 'tc_up_gs' not found

mf_plots <- plot_enrichresult(mf_enr)

## Error: object 'mf_enr' not found

mf_plots[["tree"]]

## Error: object 'mf_plots' not found

bp_enr <- tc_up_gs[["bp_enrich"]]

## Error: object 'tc_up_gs' not found

bp_plots <- plot_enrichresult(bp_enr)

## Error: object 'bp_enr' not found

bp_plots[["dot"]]

## Error: object 'bp_plots' not found

pander::pander(sessionInfo())
message(paste0("This is hpgltools commit: ", get_git_commit()))
message(paste0("Saving to ", savefile))
tmp <- sm(saveme(filename = savefile))

tmp <- loadme(filename = savefile)