ED Project: Pseudomonas RNASeq dataset.

1 Introduction

This dataset contains multiple experiments.

2 Annotations

A potentially important note, Vince just dropped in to ask about the number of genes included in the analysis. My response of ~ 5910 seemed a bit high from his perspective. I looked into the number of annotations in the Pseudomonas gff file and realized that he may prefer using the CDS instead of the gene annotations. Thus I changed the ID column when extracting the annotations to ‘Parent’ and changed the type to ‘CDS’; therefore the IDs should be the same, but limited to the set of annotations which are explicitly CDS. This lowers that number slightly to ~ 5840.

pa14_gff <- load_gff_annotations("reference/paeruginosa_pa14.gff", id_col="Parent", type="CDS")

## Trying attempt: rtracklayer::import.gff3(gff, sequenceRegionsAsSeqinfo = TRUE)

## Had a successful gff import with rtracklayer::import.gff3(gff, sequenceRegionsAsSeqinfo = TRUE)

## Returning a df with 16 columns and 5893 rows.

rownames(pa14_gff) <- pa14_gff[["Parent"]]
## The Alias column has PA14_00010

2.1 Continued concern

I spoke again with Vince and apparently this remains high, I suggested checking it against the set of PA01 annotations.

pa01_gff <- load_gff_annotations("~/libraries/genome/paeruginosa_pa01.gff", id_col="locus_tag", type="gene")

## Trying attempt: rtracklayer::import.gff3(gff, sequenceRegionsAsSeqinfo = TRUE)

## Trying attempt: rtracklayer::import.gff3(gff, sequenceRegionsAsSeqinfo = FALSE)

## Had a successful gff import with rtracklayer::import.gff3(gff, sequenceRegionsAsSeqinfo = FALSE)

## Returning a df with 15 columns and 5697 rows.

2.2 Merge gff and microbesonline annotations

pa14_microbes <- as.data.frame(load_microbesonline_annotations("PA14"))

## Found 1 entry.

## Pseudomonas aeruginosa UCBPP-PA14Proteobacteria2006-11-22yes105972208963

## The species being downloaded is: Pseudomonas aeruginosa UCBPP-PA14

## Downloading: http://www.microbesonline.org/cgi-bin/genomeInfo.cgi?tId=208963;export=tab

## The sysName column has PA14_0010

pa14_annot <- merge(pa14_gff, pa14_microbes, by.x="Alias", by.y="sysName")
rownames(pa14_annot) <- pa14_annot[["gene_id"]]

## The identifiers are a bit odd, so we need to do a little work
pa14_length <- pa14_annot[, c("gene_id", "width", "Alias")]
pa14_go <- load_microbesonline_go(species="PA14", id_column="sysName")

## Found 1 entry.

## Pseudomonas aeruginosa UCBPP-PA14Proteobacteria2006-11-22yes105972208963

## The species being downloaded is: Pseudomonas aeruginosa UCBPP-PA14 and is being downloaded as 208963.tab.

pa14_go_length <- merge(pa14_go, pa14_length, by.x="sysName", by.y="Alias")
pa14_go <- pa14_go_length[, c("gene_id", "GO")]
colnames(pa14_go) <- c("ID", "GO")
pa14_length <- pa14_go_length[, c("gene_id", "width")]
pa14_length_ids <- unique(pa14_length)[["gene_id"]]
pa14_length <- pa14_length[pa14_length_ids, ]
rownames(pa14_length) <- make.names(pa14_length[["gene_id"]], unique=TRUE)
colnames(pa14_length) <- c("ID", "width")

3 Make the expressionset

Given the above annotations, now lets pull in the counts.

I am switching to the sheet all_samples_modified_gcd.xlsx for the moment because I added a space in the strain name for the gcd sampl

pa14_expt <- create_expt("sample_sheets/all_samples_modified_gcd.xlsx",
                         gene_info=pa14_annot, file_column="hisatcounttable")

## Reading the sample metadata.

## 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.

## The sample definitions comprises: 105 rows(samples) and 31 columns(metadata fields).

## Warning in create_expt("sample_sheets/all_samples_modified_gcd.xlsx", gene_info
## = pa14_annot, : Even after changing the rownames in gene info, they do not match
## the count table.

## Even after changing the rownames in gene info, they do not match the count table.

## Here are the first few rownames from the count tables:

## gene1650835, gene1650837, gene1650839, gene1650841, gene1650843, gene1650845

## Here are the first few rownames from the gene information table:

## 

## Warning in `[.data.table`(gene_info, -columns_to_remove): Item 1 of i is -1 but
## there are only 0 rows. Ignoring this and 33 more like it out of 34.

## Bringing together the count matrix and gene information.

## Some annotations were lost in merging, setting them to 'undefined'.

## Saving the expressionset to 'expt.rda'.

## The final expressionset has 5979 rows and 105 columns.

3.1 Question: What molecule types were counted?

I have been assuming this is limited to protein coding genes, lets check…

table(fData(pa14_expt)[["source"]])

## 
## undefined 
##      5979

odd_idx <- fData(pa14_expt)[["source"]] == "undefined"
fData(pa14_expt)[odd_idx, ]

##                 Alias  seqnames   start.x       end     width  strand.x
## gene1650835 undefined undefined undefined undefined undefined undefined
## gene1650837 undefined undefined undefined undefined undefined undefined
## gene1650839 undefined undefined undefined undefined undefined undefined
## gene1650841 undefined undefined undefined undefined undefined undefined
## gene1650843 undefined undefined undefined undefined undefined undefined
## gene1650845 undefined undefined undefined undefined undefined undefined
## gene1650847 undefined undefined undefined undefined undefined undefined
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##                source      type     score     phase   gene_id      Name
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##                Dbxref    name.x    Parent     locus   locusId accession
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##                    GI scaffoldId   start.y      stop  strand.y    name.y
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##                  desc       COG    COGFun   COGDesc   TIGRFam TIGRRoles
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##                    GO        EC    ECDesc
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## gene1656272 undefined undefined undefined
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## gene1656276 undefined undefined undefined
## gene1656278 undefined undefined undefined
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## gene1656880 undefined undefined undefined
## gene1656882 undefined undefined undefined
## gene1656884 undefined undefined undefined
##  [ reached 'max' / getOption("max.print") -- omitted 2949 rows ]

4 Quick global look at the data

While we are at it, lets drop the two sad samples.

pa14_libsize <- plot_libsize(pa14_expt)
pa14_libsize$plot

pa14_nonzero <- plot_nonzero(pa14_expt)
pa14_nonzero$plot

## Warning: ggrepel: 100 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

pa14_expt <- subset_expt(pa14_expt, nonzero=5500)

## The samples (and read coverage) removed when filtering 5500 non-zero genes are:

##  SM040  SM048 
##  43984 316632

## subset_expt(): There were 105, now there are 103 samples.

4.1 A few queries

I know a priori that April is interested to see how the 4 library preparations look with respect to each other. Let us therefore create a data structure to look explicitly at that.

pa14_libprep <- set_expt_conditions(pa14_expt, fact="libraryprepbatch") %>%
  set_expt_batches(fact="organisms")

pa14_lib_norm <- normalize_expt(pa14_libprep, filter=TRUE,
                                convert="cpm", norm="quant", transform="log2")

## Removing 6 low-count genes (5973 remaining).

## transform_counts: Found 35 values equal to 0, adding 1 to the matrix.

plot_pca(pa14_lib_norm)$plot

## plot labels was not set and there are more than 100 samples, disabling it.

plot_corheat(pa14_lib_norm)$plot

libprep_pca_info <- pca_information(
    pa14_lib_norm, plot_pcas=TRUE,
    expt_factors=c("libraryprepbatch", "organisms", "strains", "media", "bioreplicate"))

## plot labels was not set and there are more than 100 samples, disabling it.

libprep_pca_info$anova_f_heatmap

libprep_pca_info$pca_plots[[2]]

## Warning: ggrepel: 76 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

libprep_pca_info$pca_plots[[3]]

## Warning: ggrepel: 102 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

libprep_pca_info$pca_plots[[4]]

## Warning: ggrepel: 85 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

libprep_pca_info$pca_plots[[5]]

## Warning: ggrepel: 77 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

To my eyes they look reasonably mixed, suggesting that library prep batch is not a dominant factor in the data.

At this point, I am thinking that we should separate the data by the experiments, but I will first just show the relationships among all the data.

5 Looking at other factors

As far as I see, there are three factors which are of primary interest:

1. PA14 strain
2. Media used
3. Bioreplicate

The last will be used as batch in the following plots.

pa14_media <- set_expt_conditions(pa14_expt, fact="media") %>%
  set_expt_batches(fact="bioreplicate")
pa14_media_norm <- normalize_expt(pa14_media, transform="log2", convert="cpm",
                                  filter=TRUE, norm="quant")

## Removing 6 low-count genes (5973 remaining).

## transform_counts: Found 35 values equal to 0, adding 1 to the matrix.

plot_pca(pa14_media_norm)$plot

## plot labels was not set and there are more than 100 samples, disabling it.

pa14_strains <- set_expt_conditions(pa14_expt, fact="strains") %>%
  set_expt_batches(fact="bioreplicate")
pa14_strains_norm <- normalize_expt(pa14_strains, transform="log2", convert="cpm",
                                  filter=TRUE, norm="quant")

## Removing 6 low-count genes (5973 remaining).
## transform_counts: Found 35 values equal to 0, adding 1 to the matrix.

plot_pca(pa14_strains_norm)$plot

## plot labels was not set and there are more than 100 samples, disabling it.

Disregarding the various experiments performed, I think we can state that media separates the data in a fashion which is more interesting than strain. Given the number of (what I assume are) closely related strains, I am thinking it might prove to be a good idea to perform my variant search tool on this data and see how well they held up with respect to the reference strain.

6 Separate the experiments

I have been told repeatedly that there are multiple experiments in this data, but apparently I have not paid proper attention because I cannot remember which is which, and to my eyes it is not obvious in the sample sheet.

With this in mind, I spoke with Solomon briefly and have an idea of the 3 logical groups in his data. Let us therefore separate and examine those first.

6.1 Metabolism and infection

For the moment, I am going to call Solomon’s samples ‘metabolism and infection.’ I will also complicate the ‘condition’ of the data by combining the media and strain, but shortly thereafter will split that back. I think the reason why will become clear.

initials_factor <- gsub(x=rownames(pData(pa14_expt)), pattern="^(..).*$", replacement="\\1")
pData(pa14_expt)[["initials"]] <- as.factor(initials_factor)
strain_media <- paste0(pData(pa14_expt)[["strains"]], "_",
                       pData(pa14_expt)[["media"]])
pData(pa14_expt)[["strain_media"]] <- strain_media

## Lets set some colors
## WT: grayscale, eda: blue, edd: green, gcd: purple, pgl: red, zwf: yellow
colors_by_strain <- list(
    "PA14 WT" = "#000000",
    "PA14 eda" = "#0000dd",
    "PA14 edd" = "#00dd00",
    "PA14 gcd" = "#dd00dd",
    "PA14 pgl" = "#dd0000",
    "PA14 zwf" = "#dddd00")

infect_metabolism <- subset_expt(pa14_expt, subset="initials=='SM'") %>%
  set_expt_conditions(fact="strains") %>%
  set_expt_batches(fact="media")

## subset_expt(): There were 103, now there are 58 samples.

plot_legend(infect_metabolism)

## $colors
##       condition           batch  colors
## sm001   PA14 WT              LB #66A61E
## sm002   PA14 WT LB + 0.5 M urea #66A61E
## sm003   PA14 WT           Urine #66A61E
## sm004   PA14 WT            PBST #66A61E
## sm005  PA14 eda           Urine #1B9E77
## sm006  PA14 eda            PBST #1B9E77
## sm007  PA14 edd           Urine #D95F02
## sm008  PA14 edd            PBST #D95F02
## sm009  PA14 gcd           Urine #7570B3
## sm010  PA14 gcd            PBST #7570B3
## sm011  PA14 pgl           Urine #E7298A
## sm012  PA14 pgl            PBST #E7298A
## sm013  PA14 zwf           Urine #E6AB02
## sm014  PA14 zwf            PBST #E6AB02
## sm015   PA14 WT              LB #66A61E
## sm016   PA14 WT LB + 0.5 M urea #66A61E
## sm017   PA14 WT           Urine #66A61E
## sm018   PA14 WT            PBST #66A61E
## sm019  PA14 eda           Urine #1B9E77
## sm020  PA14 eda            PBST #1B9E77
## sm021  PA14 edd           Urine #D95F02
## sm022  PA14 edd            PBST #D95F02
## sm023  PA14 gcd           Urine #7570B3
## sm024  PA14 gcd            PBST #7570B3
## sm025  PA14 pgl           Urine #E7298A
## sm026  PA14 pgl            PBST #E7298A
## sm027  PA14 zwf           Urine #E6AB02
## sm028  PA14 zwf            PBST #E6AB02
## sm029   PA14 WT              LB #66A61E
## sm030   PA14 WT LB + 0.5 M urea #66A61E
## sm031   PA14 WT           Urine #66A61E
## sm032   PA14 WT            PBST #66A61E
## sm033  PA14 eda           Urine #1B9E77
## sm034  PA14 eda            PBST #1B9E77
## sm035  PA14 edd           Urine #D95F02
## sm036  PA14 edd            PBST #D95F02
## sm037  PA14 gcd           Urine #7570B3
## sm038  PA14 gcd            PBST #7570B3
## sm039  PA14 pgl           Urine #E7298A
## sm041  PA14 zwf           Urine #E6AB02
## sm042  PA14 zwf            PBST #E6AB02
## sm043   PA14 WT       Instilled #66A61E
## sm044   PA14 WT       Instilled #66A61E
## sm045   PA14 WT       Instilled #66A61E
## sm046  PA14 eda       Instilled #1B9E77
## sm047  PA14 eda       Instilled #1B9E77
## sm049  PA14 edd       Instilled #D95F02
## sm050  PA14 edd       Instilled #D95F02
## sm051  PA14 edd       Instilled #D95F02
## sm052  PA14 gcd       Instilled #7570B3
## sm053  PA14 gcd       Instilled #7570B3
## sm054  PA14 gcd       Instilled #7570B3
## sm055  PA14 pgl       Instilled #E7298A
## sm056  PA14 pgl       Instilled #E7298A
## sm057  PA14 pgl       Instilled #E7298A
## sm058  PA14 zwf       Instilled #E6AB02
## sm059  PA14 zwf       Instilled #E6AB02
## sm060  PA14 zwf       Instilled #E6AB02
## 
## $plot

metabolism_control <- subset_expt(
    infect_metabolism,
    subset="media=='LB'|media=='LB + 0.5 M urea'") %>%
  set_expt_conditions(fact="media") %>%
  set_expt_batches("bioreplicate")

## subset_expt(): There were 58, now there are 6 samples.

metabolism_starvation <- subset_expt(
    infect_metabolism,
    subset="media=='PBST'|media=='Urine'") %>%
  set_expt_colors(colors=colors_by_strain) %>%
  set_expt_conditions(fact="media") %>%
  set_expt_batches(fact="strains")

## subset_expt(): There were 58, now there are 35 samples.

metabolism_starvation_strain <- set_expt_conditions(metabolism_starvation, fact="strains") %>%
  set_expt_batches(fact="media")
metabolism_exudate <- subset_expt(infect_metabolism,
                                  subset="media=='Instilled'")

## subset_expt(): There were 58, now there are 17 samples.

control_written <- write_expt(
    metabolism_control, convert="cpm", transform="raw", batch="raw", filter="raw", norm="raw",
    excel="excel/control_experiment_cpm.xlsx")

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## Loading required package: Matrix

## 
## Attaching package: 'Matrix'

## The following object is masked from 'package:S4Vectors':
## 
##     expand

## 
## Total:34 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:32 s

control_written_default <- write_expt(
    metabolism_control, batch="raw",
    excel="excel/control_experiment_defaults.xlsx")

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

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:33 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:33 s

ms_written <- write_expt(
    metabolism_starvation, convert="cpm", transform="raw", batch="raw", filter="raw", norm="raw",
    excel="excel/metabolism_starvation_experiment_cpm.xlsx")

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

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:39 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:40 s

ms_written_default <- write_expt(
    metabolism_starvation, batch="raw",
    excel="excel/metabolism_starvation_experiment_defaults.xlsx")

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

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:39 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:43 s

mss_written <- write_expt(
    metabolism_starvation_strain, convert="cpm", transform="raw", batch="raw", filter="raw", norm="raw",
    excel="excel/metabolism_starvation_strain_experiment_cpm.xlsx")

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

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:40 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:38 s

mss_written_default <- write_expt(
    metabolism_starvation_strain, batch="raw",
    excel="excel/metabolism_starvation_strain_experiment_defaults.xlsx")

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

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:38 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## 
## Total:42 s

me_written <- write_expt(
    metabolism_exudate, convert="cpm", transform="raw", batch="raw", filter="raw", norm="raw",
    excel="excel/metabolism_exudate_experiment_cpm.xlsx")

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

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## varpart sees only 1 batch, adjusting the model accordingly.

## Error in data[, colnames(data) %in% unique(all.vars(formula)), drop = FALSE] : 
##   incorrect number of dimensions

## Retrying with only condition in the model.

## 
## Total:33 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## varpart sees only 1 batch, adjusting the model accordingly.

## Error in data[, colnames(data) %in% unique(all.vars(formula)), drop = FALSE] : 
##   incorrect number of dimensions

## Retrying with only condition in the model.

## 
## Total:33 s

me_written_default <- write_expt(
    metabolism_exudate, batch="raw",
    excel="excel/metabolism_exudate_experiment_defaults.xlsx")

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

## Writing the first sheet, containing a legend and some summary data.

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## varpart sees only 1 batch, adjusting the model accordingly.

## Error in data[, colnames(data) %in% unique(all.vars(formula)), drop = FALSE] : 
##   incorrect number of dimensions

## Retrying with only condition in the model.

## 
## Total:32 s

## `geom_smooth()` using formula 'y ~ x'
## `geom_smooth()` using formula 'y ~ x'

## varpart sees only 1 batch, adjusting the model accordingly.

## Error in data[, colnames(data) %in% unique(all.vars(formula)), drop = FALSE] : 
##   incorrect number of dimensions

## Retrying with only condition in the model.

## 
## Total:32 s

6.2 Glance at these 4 subsets

As a whole group, these samples are a bit confusing. The mouse instiled samples are prety obvious, but the other sources of variance remain a bit of a mystery to me.

global_norm <- normalize_expt(infect_metabolism, filter=TRUE, convert="cpm",
                              norm="quant", transform="log2") %>%
  set_expt_conditions(fact="media")

## Removing 13 low-count genes (5966 remaining).

## transform_counts: Found 1 values equal to 0, adding 1 to the matrix.

plot_pca(global_norm)$plot

## Warning: ggrepel: 16 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

tmp <- global_norm %>%
  set_expt_conditions(fact="strains")
plot_pca(tmp)$plot

## Warning: ggrepel: 48 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

7 The smallest experiment: LB vs. LB+urea

This is a group of 6 samples, 3 in LB and three in LB+urea. This should therefore be the most straight forward comparison.

mc_norm <- normalize_expt(metabolism_control, transform="log2",
                          convert="cpm", norm="quant", filter=TRUE)

## Removing 73 low-count genes (5906 remaining).

plot_pca(mc_norm)$plot

7.1 Metabolism control experiment, DE

mc_san <- sanitize_expt(metabolism_control)
mc_de <- all_pairwise(mc_san, model_batch=TRUE, filter=TRUE)

## Plotting a PCA before surrogate/batch inclusion.

## Using limma's removeBatchEffect to visualize with(out) batch inclusion.

## Finished running DE analyses, collecting outputs.

## Comparing analyses.

mc_tables <- combine_de_tables(
    mc_de,
    excel=glue::glue("excel/metabolism_control_tables-v{ver}.xlsx"),
    sig_excel=glue::glue("excel/metabolism_control_sig-v{ver}.xlsx"))

hmm, is there any chance that SM001 and SM005 are flipped? If they were, then both clades would have 1 of each bioreplicate.

8 Starving strains

The second group is a little more complex, it seeks to simultaneously compare the strains (WT vs. mutants) and the environment (PBS vs. urine).

This design is complex enough that I think we need to choose colors more carefully.

Here is a query from Solomon:

Could you please generate me a table for the metabolism starvation strain analyses where the urine is the numerator and the PBST is the denominator?

I am interpreting this to mean we should have an experimental design in which we ignore the strains and just compare all urine and pbst samples. When I ran the following block, it looks to me that we currently return this in the following block:

ms_norm <- normalize_expt(metabolism_starvation, filter=TRUE, norm="quant",
                          convert="cpm", transform="log2")

## Removing 30 low-count genes (5949 remaining).

plot_pca(ms_norm)$plot

ms_de <- all_pairwise(metabolism_starvation, model_batch=TRUE)

## Plotting a PCA before surrogate/batch inclusion.

## Using limma's removeBatchEffect to visualize with(out) batch inclusion.

## Finished running DE analyses, collecting outputs.

## Comparing analyses.

ms_de$comparison$comp

##                Urine_vs_PBST
## limma_vs_deseq        0.9830
## limma_vs_edger        0.9829
## limma_vs_ebseq        0.9772
## limma_vs_basic        0.9996
## deseq_vs_edger        0.9999
## deseq_vs_ebseq        0.9953
## deseq_vs_basic        0.9833
## edger_vs_ebseq        0.9963
## edger_vs_basic        0.9833
## ebseq_vs_basic        0.9810

ms_tables <- combine_de_tables(
    ms_de,
    excel=glue::glue("excel/metabolism_starvation_tables-v{ver}.xlsx"),
    sig_excel=glue::glue("excel/metabolism_starvation_sig-v{ver}.xlsx"))

9 Metabolism Starvation GO

ms_up <- ms_tables[["significant"]][["deseq"]][["ups"]][[1]]
ms_down <- ms_tables[["significant"]][["deseq"]][["downs"]][[1]]

## The go data from microbesonline is keyed by the gene name
## (e.g. dnaA), not gene ID or PA id or whatever.
pa14_lengths <- pa14_annot[, c("name.x", "width")]
colnames(pa14_lengths) <- c("ID", "width")

rownames(ms_up) <- make.names(ms_up[["namex"]], unique=TRUE)
ms_up_goseq <- simple_goseq(ms_up, go_db=pa14_go, length_db=pa14_lengths)

## Found 0 go_db genes and 0 length_db genes out of 640.

## Error in if (hi <= low) {: missing value where TRUE/FALSE needed

ms_up_goseq[["pvalue_plots"]][["bpp_plot_over"]]

## Error in eval(expr, envir, enclos): object 'ms_up_goseq' not found

ms_up_goseq[["pvalue_plots"]][["mfp_plot_over"]]

## Error in eval(expr, envir, enclos): object 'ms_up_goseq' not found

rownames(ms_down) <- make.names(ms_down[["namex"]], unique=TRUE)
ms_down_goseq <- simple_goseq(ms_down, go_db=pa14_go, length_db=pa14_lengths)

## Found 0 go_db genes and 0 length_db genes out of 679.

## Error in if (hi <= low) {: missing value where TRUE/FALSE needed

ms_down_goseq[["pvalue_plots"]][["bpp_plot_over"]]

## Error in eval(expr, envir, enclos): object 'ms_down_goseq' not found

ms_down_goseq[["pvalue_plots"]][["mfp_plot_over"]]

## Error in eval(expr, envir, enclos): object 'ms_down_goseq' not found

9.1 Compare strains

This time let us compare the strains and lower the variance from media.

mss_norm <- normalize_expt(metabolism_starvation_strain, filter=TRUE, convert="cpm", norm="quant",
                           transform="log2")

## Removing 30 low-count genes (5949 remaining).

plot_pca(mss_norm)$plot

mss_urine <- subset_expt(metabolism_starvation_strain, subset="batch=='Urine'")

## subset_expt(): There were 35, now there are 18 samples.

mss_urine_norm <- normalize_expt(mss_urine, filter=TRUE, convert="cpm", norm="quant",
                                 batch="svaseq", transform="log2")

## Warning in normalize_expt(mss_urine, filter = TRUE, convert = "cpm", norm =
## "quant", : Quantile normalization and sva do not always play well together.

## Removing 38 low-count genes (5941 remaining).

## batch_counts: Before batch/surrogate estimation, 0 entries are x==0: 0%.

## batch_counts: Before batch/surrogate estimation, 537 entries are 0<x<1: 1%.

## Setting 5 low elements to zero.

## transform_counts: Found 5 values equal to 0, adding 1 to the matrix.

plot_pca(mss_urine_norm)$plot

mss_pbst <- subset_expt(metabolism_starvation_strain, subset="batch=='PBST'")

## subset_expt(): There were 35, now there are 17 samples.

mss_pbst_norm <- normalize_expt(mss_pbst, filter=TRUE, convert="cpm",
                                batch="svaseq", transform="log2")

## Removing 57 low-count genes (5922 remaining).

## batch_counts: Before batch/surrogate estimation, 68 entries are x==0: 0%.

## batch_counts: Before batch/surrogate estimation, 1068 entries are 0<x<1: 1%.

## Setting 14 low elements to zero.

## transform_counts: Found 14 values equal to 0, adding 1 to the matrix.

plot_pca(mss_pbst_norm)$plot

interesting <- list(
    "eda_vs_wt" = c("PA14eda", "PA14WT"),
    "edd_vs_wt" = c("PA14edd", "PA14WT"),
    "gcd_vs_wt" = c("PA14gcd", "PA14WT"),
    "pgl_vs_wt" = c("PA14pgl", "PA14WT"),
    "zfw_vs_wt" = c("PA14zwf", "PA14WT"))
mss_urine_de <- all_pairwise(mss_urine, model_batch="svaseq", filter=TRUE)

## batch_counts: Before batch/surrogate estimation, 29 entries are x==0: 0%.

## Plotting a PCA before surrogate/batch inclusion.

## Using svaseq to visualize before/after batch inclusion.

## Performing a test normalization with: raw

## Removing 0 low-count genes (5941 remaining).

## batch_counts: Before batch/surrogate estimation, 29 entries are x==0: 0%.

## batch_counts: Before batch/surrogate estimation, 506 entries are 0<x<1: 0%.

## Setting 11 low elements to zero.

## transform_counts: Found 11 values equal to 0, adding 1 to the matrix.

## Finished running DE analyses, collecting outputs.

## Comparing analyses.

mss_urine_table <- combine_de_tables(
    mss_urine_de, keepers=interesting,
    excel=glue::glue("excel/metabolism_starvation_strain_tables-v{ver}.xlsx"),
    sig_excel=glue::glue("excel/metabolism_starvation_strain_sig-v{ver}.xlsx"))

Given that the strains are so similar, we can comfortably compare them across media (PBST/urine).

msm <- set_expt_conditions(metabolism_starvation_strain, fact="batch") %>%
  set_expt_batches(fact="bioreplicate")
msm_norm <- normalize_expt(msm, filter=TRUE, convert="cpm", norm="quant",
                           transform="log2")

## Removing 30 low-count genes (5949 remaining).

plot_pca(msm_norm)$plot

10 Exudate

Compare the strains during the instillation process. Note that I think Vince wants to use a variant of one of these images for a grant, and wants to see the confidence intervals; so I am removing the eda strain because it has only two samples.

exudate_norm <- normalize_expt(metabolism_exudate, filter=TRUE, convert="cpm",
                               norm="quant", transform="log2")

## Removing 69 low-count genes (5910 remaining).

## transform_counts: Found 76 values equal to 0, adding 1 to the matrix.

pp(file="images/compare_strains_exudate.pdf", image=plot_pca(exudate_norm)$plot)

exudate_nb <- normalize_expt(metabolism_exudate, filter=TRUE, convert="cpm",
                             transform="log2", batch="svaseq")

## Removing 69 low-count genes (5910 remaining).

## batch_counts: Before batch/surrogate estimation, 1605 entries are x==0: 2%.

## batch_counts: Before batch/surrogate estimation, 2460 entries are 0<x<1: 2%.

## Setting 393 low elements to zero.

## transform_counts: Found 393 values equal to 0, adding 1 to the matrix.

plot_pca(exudate_nb)$plot

pp(file="images/compare_strains_exudate_sva.pdf", image=plot_pca(exudate_nb)$plot)

exudate_noeda <- subset_expt(metabolism_exudate, subset="condition!='PA14 eda'")

## subset_expt(): There were 17, now there are 15 samples.

10.1 Exudate DE

exudate_de <- all_pairwise(metabolism_exudate, model_batch=TRUE, filter=TRUE)

## Plotting a PCA before surrogate/batch inclusion.

## Using limma's removeBatchEffect to visualize with(out) batch inclusion.

## Finished running DE analyses, collecting outputs.

## Comparing analyses.

exudate_tables <- combine_de_tables(
    exudate_de, keepers=interesting,
    excel=glue::glue("excel/exudate_tables-v{ver}.xlsx"),
    sig_excel=glue::glue("excel/exudate_sig-v{ver}.xlsx"))

pa_pgl_wanted_table <- exudate_tables[["data"]][["pgl_vs_wt"]]
pa_pgl_wt_volcano <- plot_volcano_de(pa_pgl_wanted_table, logfc=2, fc_col="deseq_logfc", p_col="deseq_adjp")
## 45 ds, 715fci, 5135pi, 15us -> 5910
pp(file="images/wt_vs_pgl_de_volcano_instilled_paeruginosa.pdf",
   image=pa_pgl_wt_volcano$plot)

dev.off()

## png 
##   3

11 Instilled vs. PBST/Urine

Vince and Najib are interested in a slightly different question:

instilled_vs <- subset_expt(
    pa14_expt,
    subset='media=="PBST"|media=="Urine"|media=="Instilled"') %>%
  set_expt_conditions(fact="media") %>%
  set_expt_batches(fact="bioreplicate")

## subset_expt(): There were 103, now there are 52 samples.

instilled_vs_norm <- normalize_expt(instilled_vs, transform="log2", convert="cpm",
                                    norm="quant", filter=TRUE)

## Removing 13 low-count genes (5966 remaining).

## transform_counts: Found 1 values equal to 0, adding 1 to the matrix.

inst_pca <- plot_pca(instilled_vs_norm)$plot
pp(file="images/instilled_vs_pca.pdf", image=inst_pca)

## Warning: ggrepel: 10 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

## Warning: ggrepel: 10 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

inst_vs_de <- all_pairwise(instilled_vs, filter=TRUE)

## Plotting a PCA before surrogate/batch inclusion.

## Using limma's removeBatchEffect to visualize with(out) batch inclusion.

## Finished running DE analyses, collecting outputs.

## Comparing analyses.

inst_vs_tables <- combine_de_tables(inst_vs_de, excel="excel/instilled_vs_tables.xlsx")

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

inst_table <- inst_vs_tables[["data"]][["Urine_vs_Instilled"]]
urine_inst_pa_volcano <- plot_volcano_de(inst_table, logfc=1,
                                         fc_col="deseq_logfc", p_col="deseq_adjp")
pp(file="images/pbst_vs_instilled_de_volcano.pdf",
   image=inst_vs_tables[["plots"]][["PBST_vs_Instilled"]][["deseq_vol_plots"]][["plot"]])

12 Check mouse counts

mm_annot <- load_biomart_annotations(species="mmusculus")[["annotation"]]

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

rownames(mm_annot) <- paste0("gene:", make.names(mm_annot[["ensembl_gene_id"]], unique=TRUE))
mm_expt <- create_expt("sample_sheets/all_samples_modified2.xlsx",
                       gene_info=mm_annot, file_column="mousetable") %>%
  set_expt_conditions(fact="strains") %>%
  set_expt_batches(fact="bioreplicate") %>%
  subset_expt(subset="media=='Mice instiled'")

## Reading the sample metadata.

## 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.

## The sample definitions comprises: 105 rows(samples) and 32 columns(metadata fields).

## Warning in create_expt("sample_sheets/all_samples_modified2.xlsx", gene_info =
## mm_annot, : Some samples were removed when cross referencing the samples against
## the count data.

## Matched 25654 annotations and counts.

## Bringing together the count matrix and gene information.

## Some annotations were lost in merging, setting them to 'undefined'.

## Warning in create_expt("sample_sheets/all_samples_modified2.xlsx", gene_info =
## mm_annot, : The following samples have no counts! SM029SM032SM038SM040

## Saving the expressionset to 'expt.rda'.

## The final expressionset has 25753 rows and 35 columns.

## subset_expt(): There were 35, now there are 18 samples.

plot_libsize(mm_expt)$plot

mm_norm <- normalize_expt(mm_expt, transform="log2", convert="cpm", filter=TRUE, norm="quant")

## Removing 12222 low-count genes (13531 remaining).

## transform_counts: Found 1459 values equal to 0, adding 1 to the matrix.

plot_pca(mm_norm)$plot

mm_v2 <- subset_expt(mm_expt, subset="condition=='PA14 WT'|condition=='PA14 pgl'")

## subset_expt(): There were 18, now there are 6 samples.

mm_de <- all_pairwise(mm_v2, model_batch=TRUE)

## Plotting a PCA before surrogate/batch inclusion.

## Using limma's removeBatchEffect to visualize with(out) batch inclusion.

## Finished running DE analyses, collecting outputs.

## Comparing analyses.

keeper <- list(
    "pgl_vs_wt" = c("PA14pgl", "PA14WT"))
mm_table <- combine_de_tables(mm_de, keepers=keeper)
mm_wanted_table <- mm_table[["data"]][["pgl_vs_wt"]]
pgl_mm_volcano <- plot_volcano_de(mm_wanted_table, logfc=1, fc_col="deseq_logfc", p_col="deseq_adjp")
## ds:23, us:9, fci:37, 25684
pp(file="images/wt_vs_pgl_de_volcano_instilled_mmusculus.pdf",
   image=pgl_mm_volcano$plot)

dev.off()

## png 
##   3

13 Fun with circos

control_table <- mc_tables[["data"]][["LB05Murea_vs_LB"]]
urine_eda <- mss_urine_table[["data"]][["eda_vs_wt"]]
urine_edd <- mss_urine_table[["data"]][["edd_vs_wt"]]
urine_gcd <- mss_urine_table[["data"]][["gcd_vs_wt"]]
urine_pgl <- mss_urine_table[["data"]][["pgl_vs_wt"]]
urine_zfw <- mss_urine_table[["data"]][["zfw_vs_wt"]]

exudate_eda <- exudate_tables[["data"]][["eda_vs_wt"]]
exudate_edd <- exudate_tables[["data"]][["edd_vs_wt"]]
exudate_gcd <- exudate_tables[["data"]][["gcd_vs_wt"]]
exudate_pgl <- exudate_tables[["data"]][["pgl_vs_wt"]]
exudate_zfw <- exudate_tables[["data"]][["zfw_vs_wt"]]

pa14_annot[["chromosome"]] <- "Pseudomonas_aeruginosa_UCBPP_PA14"

## Error in `[[<-.data.frame`(`*tmp*`, "chromosome", value = "Pseudomonas_aeruginosa_UCBPP_PA14"): replacement has 1 row, data has 0

sm_cfg <- circos_prefix(pa14_annot, name="sm", cog_column = "COGFun",
                        start_column="start.x", end_column="end", strand_column="strand.x",
                        chr_column="chromosome", id_column="gene_id")

## This assumes you have a colors.conf in circos/colors/ and fonts.conf in circos/fonts/

## It also assumes you have conf/ideogram.conf, conf/ticks.conf, and conf/housekeeping.conf

## It will write circos/conf/sm.conf with a reasonable first approximation config file.

## Error in circos_prefix(pa14_annot, name = "sm", cog_column = "COGFun", : This function requires some way of understanding plus/minus strand.

sm_kary <- circos_karyotype(sm_cfg, fasta="reference/paeruginosah_pa14.fasta")

## Error in circos_karyotype(sm_cfg, fasta = "reference/paeruginosah_pa14.fasta"): object 'sm_cfg' not found

sm_plus_minus <- circos_plus_minus(sm_cfg, width=0.06, thickness=40)

## Error in circos_plus_minus(sm_cfg, width = 0.06, thickness = 40): object 'sm_cfg' not found

## Put the plots here
sm_first_heat <- circos_heatmap(sm_cfg, control_table, colname="deseq_logfc",
                                basename="control", outer=sm_plus_minus, width=0.05)

## Error in circos_heatmap(sm_cfg, control_table, colname = "deseq_logfc", : object 'sm_cfg' not found

##sm_eda_hist <- circos_hist(sm_cfg, urine_eda, colname="deseq_logfc",
##                           basename="ureda", outer=sm_first_hist, spacing=-0.05)
##sm_edd_hist <- circos_hist(sm_cfg, urine_edd, colname="deseq_logfc",
##                           basename="uredd", outer=sm_eda_hist, spacing=-0.05)
##sm_gcd_hist <- circos_hist(sm_cfg, urine_gcd, colname="deseq_logfc",
##                           basename="urgcd", outer=sm_edd_hist, spacing=-0.05)
##sm_pgl_hist <- circos_hist(sm_cfg, urine_pgl, colname="deseq_logfc",
##                           basename="urpgl", outer=sm_gcd_hist, spacing=-0.05)
##sm_zfw_hist <- circos_hist(sm_cfg, urine_zfw, colname="deseq_logfc",
##                           basename="urzfw", outer=sm_pgl_hist, spacing=-0.05)
##ex_eda_hist <- circos_hist(sm_cfg, exudate_eda, colname="deseq_logfc",
##                           basename="exeda", outer=sm_zfw_hist, spacing=-0.05)
##ex_edd_hist <- circos_hist(sm_cfg, exudate_edd, colname="deseq_logfc",
##                           basename="exedd", outer=ex_eda_hist, spacing=-0.05)
##ex_gcd_hist <- circos_hist(sm_cfg, exudate_gcd, colname="deseq_logfc",
##                           basename="exgcd", outer=ex_edd_hist, spacing=-0.05)
##ex_pgl_hist <- circos_hist(sm_cfg, exudate_pgl, colname="deseq_logfc",
##                           basename="expgl", outer=ex_gcd_hist, spacing=-0.05)
##ex_zfw_hist <- circos_hist(sm_cfg, exudate_zfw, colname="deseq_logfc",
##                           basename="exzfw", outer=ex_pgl_hist, spacing=-0.05)
sm_finish <- circos_suffix(sm_cfg)

## Error in circos_suffix(sm_cfg): object 'sm_cfg' not found

sm_made <- circos_make(sm_cfg, target="sm")

## Error in dirname(cfg@data_dir): object 'sm_cfg' not found

pander::pander(sessionInfo())
message(paste0("This is hpgltools commit: ", get_git_commit()))
this_save <- paste0(gsub(pattern="\\.Rmd", replace="", x=rmd_file), "-v", ver, ".rda.xz")
message(paste0("Saving to ", this_save))
tmp <- sm(saveme(filename=this_save))