M.tuberculosis 20180913: Preprocessing DIA data with two libraries.
OpenSwathWorkflow against the Tuberculist transitions
The following invocations of OpenMS/pyprophet/tric are included in:
This script includes the process for generating a local, comet-based transition library from DDA samples generated locally as well as the steps used when processing the downloaded transition libraries.
Gathering parameters
In this first block, I will set a couple of variables and source a file containing the parameters for the rest of the script.
Plotting metrics of the raw data
The raw data files provide opportunities to make sure that the later invocations of openMS/etc will actually work; for example, if there are too few transitions observed here, one should not be surprised if pyprophet and tric fail later.
sample_sheet <- "sample_sheets/Mtb_dia_samples_20180913.xlsx"
savefile <- "mzxml_dia_data_20180913.rda"
metadata <- openxlsx::read.xlsx(sample_sheet)
keeper_idx <- !grepl(pattern="^#", x=metadata[["sampleid"]])
metadata <- metadata[keeper_idx, ]
knitr::kable(metadata)| sampleid | TubeID | tubelabel | FigureReplicate | Figure.Name | Sample.Description | Bio.Replicate | LC.Run.(tentative.certainly.wrong.until.I.bug.Yan) | MS.Run.(tentative.wrong.until.I.bug.Yan) | Technical.Replicate | Replicate.State | Run | expt_id | Genotype | Collection.Type | Condition | batch | windowsize | enzyme | harvestdate | prepdate | rundate | runinfo | rawfile | Filename | dia_scored | tuberculist_scored | include_exclude | Run_note |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 2018_0817BrikenTrypsinDIA01 | 20180404.07 | 4.4.18 07-7 | 01 | delta_filtrate_01 | H37Rv ΔEsx-5A; Culture Filtrate | br01 | 20180828 | 01 | tr06 | br01_tr06 | 2018_0817BrikenTrypsinDIA01 | aug2018 | delta | filtrate | delta_filtrate | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA01.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA01.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA01_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA01_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA02 | 20180404.08 | 4.4.18 07-8 | 02 | delta_filtrate_02 | H37Rv ΔEsx-5A; Culture Filtrate | br02 | 20180828 | 01 | tr06 | br02_tr06 | 2018_0817BrikenTrypsinDIA02 | aug2018 | delta | filtrate | delta_filtrate | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA02.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA02.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA02_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA02_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA03 | 20180404.09 | 4.4.18 07-9 | 03 | delta_filtrate_03 | H37Rv ΔEsx-5A; Culture Filtrate | br03 | 20180828 | 01 | tr06 | br03_tr06 | 2018_0817BrikenTrypsinDIA03 | aug2018 | delta | filtrate | delta_filtrate | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA03.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA03.mzXML | NA | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA03_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA07 | 20180215.04 | 2.15.18-4 | 01 | wt_filtrate_01 | WT H37Rv Culture Filtrate | br07 | 20180828 | 01 | tr06 | br07_tr06 | 2018_0817BrikenTrypsinDIA07 | aug2018 | wt | filtrate | wt_filtrate | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA07.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA07.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA07_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA07_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA08 | 20180215.05 | 2.15.18-5 | 02 | wt_filtrate_02 | WT H37Rv Culture Filtrate | br08 | 20180828 | 01 | tr06 | br08_tr06 | 2018_0817BrikenTrypsinDIA08 | aug2018 | wt | filtrate | wt_filtrate | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA08.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA08.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA08_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA08_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA09 | 20180215.06 | 2.15.18-6 | 03 | wt_filtrate_03 | WT H37Rv Culture Filtrate | br09 | 20180828 | 01 | tr06 | br09_tr06 | 2018_0817BrikenTrypsinDIA09 | aug2018 | wt | filtrate | wt_filtrate | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA09.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA09.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA09_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA09_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA11 | 20180404.01 | 4.4.18 07-1 | 01 | delta_whole_01 | H37Rv ΔEsx-5A; Whole Cell Lysate | br10 | 20180828 | 01 | tr06 | br10_tr06 | 2018_0817BrikenTrypsinDIA11 | aug2018 | delta | whole | delta_whole | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA11.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA11.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA11_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA11_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA12 | 20180404.02 | 4.4.18 07-2 | 02 | delta_whole_02 | H37Rv ΔEsx-5A; Whole Cell Lysate | br11 | 20180828 | 01 | tr06 | br11_tr06 | 2018_0817BrikenTrypsinDIA12 | aug2018 | delta | whole | delta_whole | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA12.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA12.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA12_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA12_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA13 | 20180404.03 | 4.4.18 07-3 | 03 | delta_whole_03 | H37Rv ΔEsx-5A; Whole Cell Lysate | br12 | 20180828 | 01 | tr06 | br12_tr06 | 2018_0817BrikenTrypsinDIA13 | aug2018 | delta | whole | delta_whole | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA13.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA13.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA13_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA13_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA17 | 20180215.01 | 2.15.18-1 | 01 | wt_whole_01 | WT H37Rv Whole Cell Lysate | br16 | 20180828 | 01 | tr06 | br16_tr06 | 2018_0817BrikenTrypsinDIA17 | aug2018 | wt | whole | wt_whole | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA17.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA17.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA17_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA17_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA18 | 20180215.02 | 2.15.18-2 | 02 | wt_whole_02 | WT H37Rv Whole Cell Lysate | br17 | 20180828 | 01 | tr06 | br17_tr06 | 2018_0817BrikenTrypsinDIA18 | aug2018 | wt | whole | wt_whole | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA18.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA18.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA18_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA18_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
| 2018_0817BrikenTrypsinDIA19 | 20180215.03 | 2.15.18-3 | 03 | wt_whole_03 | WT H37Rv Whole Cell Lysate | br18 | 20180828 | 01 | tr06 | br18_tr06 | 2018_0817BrikenTrypsinDIA19 | aug2018 | wt | whole | wt_whole | aug2018 | 8 | Trypsin | 20171019 | 20180828 | 20180717 | 8 m/z SWATH windows | results/00raw/20180828/2018_0817BrikenTrypsinDIA19.raw | results/01mzXML/dia/20180817/2018_0817BrikenTrypsinDIA19.mzXML | results/08pyprophet/20180817/whole_8mz/2018_0817BrikenTrypsinDIA19_vs_20180817_whole_HCD_dia_scored.tsv | results/08pyprophet/20180817/whole_8mz_tuberculist/2018_0817BrikenTrypsinDIA19_vs_20180817_whole_HCD_dia_scored.tsv | NA | NA |
tmpidx <- grep(pattern="2018_0817", x=metadata[["sampleid"]])
metadata <- metadata[tmpidx, ]
if (file.exists(savefile)) {
load(savefile)
} else {
mzxml_data <- extract_mzxml_data(metadata,
file_column="Filename",
savefile=savefile)
}
intensity_boxplot <- plot_mzxml_boxplot(mzxml_data)## Adding 2018_0817BrikenTrypsinDIA01## Adding 2018_0817BrikenTrypsinDIA02## Adding 2018_0817BrikenTrypsinDIA03## Adding 2018_0817BrikenTrypsinDIA07## Adding 2018_0817BrikenTrypsinDIA08## Adding 2018_0817BrikenTrypsinDIA09## Adding 2018_0817BrikenTrypsinDIA11## Adding 2018_0817BrikenTrypsinDIA12## Adding 2018_0817BrikenTrypsinDIA13## Adding 2018_0817BrikenTrypsinDIA17## Adding 2018_0817BrikenTrypsinDIA18## Adding 2018_0817BrikenTrypsinDIA19## This data will benefit from being displayed on the log scale.## If this is not desired, set scale='raw'## Some entries are 0. We are on log scale, adding 1 to the data.## Changed 176131 zero count features.## Writing the image to: images/20180913_dia_mzxml_intensities-v20180913.png and calling dev.off().
## Adding 2018_0817BrikenTrypsinDIA01## Adding 2018_0817BrikenTrypsinDIA02## Adding 2018_0817BrikenTrypsinDIA03## Adding 2018_0817BrikenTrypsinDIA07## Adding 2018_0817BrikenTrypsinDIA08## Adding 2018_0817BrikenTrypsinDIA09## Adding 2018_0817BrikenTrypsinDIA11## Adding 2018_0817BrikenTrypsinDIA12## Adding 2018_0817BrikenTrypsinDIA13## Adding 2018_0817BrikenTrypsinDIA17## Adding 2018_0817BrikenTrypsinDIA18## Adding 2018_0817BrikenTrypsinDIA19## Writing the image to: images/20180913_dia_mzxml_retention-v20180913.png and calling dev.off().
## Adding 2018_0817BrikenTrypsinDIA01## Adding 2018_0817BrikenTrypsinDIA02## Adding 2018_0817BrikenTrypsinDIA03## Adding 2018_0817BrikenTrypsinDIA07## Adding 2018_0817BrikenTrypsinDIA08## Adding 2018_0817BrikenTrypsinDIA09## Adding 2018_0817BrikenTrypsinDIA11## Adding 2018_0817BrikenTrypsinDIA12## Adding 2018_0817BrikenTrypsinDIA13## Adding 2018_0817BrikenTrypsinDIA17## Adding 2018_0817BrikenTrypsinDIA18## Adding 2018_0817BrikenTrypsinDIA19## Writing the image to: images/20180913_dia_mzxml_mzbase-v20180913.png and calling dev.off().
## Adding 2018_0817BrikenTrypsinDIA01## Adding 2018_0817BrikenTrypsinDIA02## Adding 2018_0817BrikenTrypsinDIA03## Adding 2018_0817BrikenTrypsinDIA07## Adding 2018_0817BrikenTrypsinDIA08## Adding 2018_0817BrikenTrypsinDIA09## Adding 2018_0817BrikenTrypsinDIA11## Adding 2018_0817BrikenTrypsinDIA12## Adding 2018_0817BrikenTrypsinDIA13## Adding 2018_0817BrikenTrypsinDIA17## Adding 2018_0817BrikenTrypsinDIA18## Adding 2018_0817BrikenTrypsinDIA19## Writing the image to: images/20180913_dia_mzxml_scanintensity-v20180913.png and calling dev.off().
Converting downloaded transitions
In block 15 of dia_invocation_20180913.sh the command used is prested and copied here for interactive running.
echo "Converting the Tuberculist libraries to pqp."
TUBERCULIST_TRAML="results/05spectral_libraries/Mtb_TubercuList-R27_iRT_UPS_noMox_noMC_sall_osw_decoy.TraML"
echo "The input is: ${TUBERCULIST_TRAML}"
echo "The output is: ${TUBERCULIST_PQP}"
TargetedFileConverter \
-in "${TUBERCULIST_TRAML}" \
-in_type TraML \
-out "${TUBERCULIST_PQP}" \
-out_type pqp \
2>"${TUBERCULIST_PQP}_convert.log" 1>&2OpenSwathWorkflow invocation
block 16 contains the commands used to run openswathworkflow and pyprophet. Those are repeated here in order to test them interactively when needed.
echo "Invoking the OpenSwathWorkflow using the tuberculist transitions."
base_mzxmldir="results/01mzXML/dia/${VERSION}"
swath_inputs=$(/bin/ls "${base_mzxmldir}")
echo "Checking in, the inputs are: ${swath_inputs}"
mkdir -p "${TUBERCULIST_OUTDIR}"
pypdir="${PYPROPHET_OUTDIR}_tuberculist"
mkdir -p "${pypdir}"
for input in ${swath_inputs}
do
in_mzxml="${base_mzxmldir}/${input}"
name=$(basename "${input}" .mzXML)
echo "Starting openswath run of ${name} using ${MZ_WINDOWS} windows at $(date)."
tb_output_prefix="${TUBERCULIST_OUTDIR}/${name}_vs_${VERSION}_${TYPE}_${DDA_METHOD}_dia"
pyprophet_output_prefix="${pypdir}/${name}_vs_${VERSION}_${TYPE}_${DDA_METHOD}_dia"
echo "Deleting previous swath output file: ${tb_output_prefix}.osw"
rm -f "${tb_output_prefix}.osw"
OpenSwathWorkflow \
-ini "parameters/openms_${VERSION}.ini" \
-in "${in_mzxml}" \
-swath_windows_file "windows/openswath_${name}.txt" \
-tr "${TUBERCULIST_PQP}" \
-out_osw "${tb_output_prefix}.osw" \
2>"${tb_output_prefix}_osw.log" 1>&2
if [[ "$?" -ne "0" ]]; then
echo "OpenSwathWorkflow for ${name} failed."
fi
rm -f "${tb_output_prefix}_scored.osw"
echo "Scoring individual swath run: ${tb_output_prefix}"
pyprophet \
score \
--level ms1 \
--in "${tb_output_prefix}.osw" \
--out "${pyprophet_output_prefix}_scored.osw" \
2>>"${pyprophet_output_prefix}_pyprophet_ms1.log" 1>&2
if [[ "$?" -ne "0" ]]; then
echo "MS1 scoring ${pyprophet_output_prefix}_scored.osw failed."
fi
pyprophet \
score \
--level ms2 \
--in "${pyprophet_output_prefix}_scored.osw" \
--out "${pyprophet_output_prefix}_scored.osw" \
2>>"${pyprophet_output_prefix}_pyprophet_ms2.log" 1>&2
if [[ "$?" -ne "0" ]]; then
echo "MS2 scoring ${pyprophet_output_prefix}_scored.osw failed."
fi
pyprophet \
protein \
--in "${pyprophet_output_prefix}_scored.osw" \
--context run-specific \
2>>"${pyprophet_output_prefix}_pyprophet_protein.log" 1>&2
if [[ "$?" -ne "0" ]]; then
echo "Protein scoring ${pyprophet_output_prefix}_scored.osw failed."
fi
rm -f "${pyprophet_output_prefix}_scored.tsv"
echo "Exporting individual swath run: to ${pyprophet_output_prefix}_scored.tsv"
pyprophet \
export \
--in "${pyprophet_output_prefix}_scored.osw" \
--out "${pyprophet_output_prefix}_scored.tsv" \
2>>"${pyprophet_output_prefix}_pyprophet_export.log" 1>&2
## ok something is fubar, the stupid tsv files are being written in the cwd as run_filename.tsv
## No matter what I do!
mv "${input}.tsv" "${pyprophet_output_prefix}_scored.tsv"
if [[ "$?" -ne "0" ]]; then
echo "Exporting ${pyprophet_output_prefix}_scored.tsv failed."
fi
doneMerging the Tuberculist-derived data with TRIC
Finally, block 17 of the invocation script provides the command used to make the final, feature-aligned data which is used by SWATH2stats and friends. In addition, it generates a matrix of intensities by sample and some metadata. Once again, it is copy/pasted here to allow interactive testing.
tric_tb="${TRIC_OUTDIR}_tuberculist"
mkdir -p "${tric_tb}"
feature_alignment.py \
--force \
--in "./${pypdir}/"*.tsv \
--out "${tric_tb}/${SEARCH_METHOD}_${DDA_METHOD}.tsv" \
--out_matrix "${tric_tb}/${DDA_METHOD}_outmatrix.tsv" \
--out_meta "${tric_tb}/${DDA_METHOD}_meta.tsv"
2>"${tric_tb}/feature_alignment.err" \
1>"${tric_tb}/feature_alignment.out"
echo "Wrote final output to ${tric_tb}/${SEARCH_METHOD}_${DDA_METHOD}.tsv"Index version: 20180913
TODO
- 2017-06-14:
if (!isTRUE(get0("skip_load"))) {
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))
pander::pander(sessionInfo())
}## If you wish to reproduce this exact build of hpgltools, invoke the following:## > git clone http://github.com/abelew/hpgltools.git## > git reset f791b304824ce2290b018f28c7495bc9b4af9b38## This is hpgltools commit: Thu Oct 25 15:57:52 2018 -0400: f791b304824ce2290b018f28c7495bc9b4af9b38## Saving to 01_preprocessing_comet_20180806-v20180913.rda.xzR version 3.5.1 (2018-07-02)
Platform: x86_64-pc-linux-gnu (64-bit)
locale: LC_CTYPE=en_US.utf8, LC_NUMERIC=C, LC_TIME=en_US.utf8, LC_COLLATE=en_US.utf8, LC_MONETARY=en_US.utf8, LC_MESSAGES=en_US.utf8, LC_PAPER=en_US.utf8, LC_NAME=C, LC_ADDRESS=C, LC_TELEPHONE=C, LC_MEASUREMENT=en_US.utf8 and LC_IDENTIFICATION=C
attached base packages: stats, graphics, grDevices, utils, datasets, methods and base
other attached packages: foreach(v.1.4.4) and hpgltools(v.2018.03)
loaded via a namespace (and not attached): tidyselect(v.0.2.5), xfun(v.0.3), pander(v.0.6.2), purrr(v.0.2.5), doSNOW(v.1.0.16), colorspace(v.1.3-2), snow(v.0.4-3), miniUI(v.0.1.1.1), htmltools(v.0.3.6), yaml(v.2.2.0), base64enc(v.0.1-3), rlang(v.0.3.0), later(v.0.7.5), pillar(v.1.3.0), glue(v.1.3.0), withr(v.2.1.2), RColorBrewer(v.1.1-2), BiocGenerics(v.0.26.0), bindrcpp(v.0.2.2), questionr(v.0.6.3), plyr(v.1.8.4), bindr(v.0.1.1), stringr(v.1.3.1), munsell(v.0.5.0), commonmark(v.1.6), gtable(v.0.2.0), zip(v.1.0.0), devtools(v.1.13.6), codetools(v.0.2-15), evaluate(v.0.12), memoise(v.1.1.0), Biobase(v.2.40.0), knitr(v.1.20), rmdformats(v.0.3.3), doParallel(v.1.0.14), httpuv(v.1.4.5), parallel(v.3.5.1), highr(v.0.7), Rcpp(v.0.12.19), xtable(v.1.8-3), promises(v.1.0.1), scales(v.1.0.0), backports(v.1.1.2), mime(v.0.6), ggplot2(v.3.0.0), digest(v.0.6.18), openxlsx(v.4.1.0), stringi(v.1.2.4), bookdown(v.0.7), dplyr(v.0.7.7), shiny(v.1.1.0), grid(v.3.5.1), rprojroot(v.1.3-2), tools(v.3.5.1), magrittr(v.1.5), lazyeval(v.0.2.1), tibble(v.1.4.2), crayon(v.1.3.4), pkgconfig(v.2.0.2), xml2(v.1.2.0), data.table(v.1.11.8), assertthat(v.0.2.0), rmarkdown(v.1.10), roxygen2(v.6.1.0), rstudioapi(v.0.8), iterators(v.1.0.10), R6(v.2.3.0) and compiler(v.3.5.1)