RNAseq of T.cruzi CL-Brener and CL-14.

1 Outline

There are a few steps involved in this process:

• preprocessing.html Performing sequence trimming, estimation, mapping, and counting.
• annotation.html Data shared across analyses (annotations, genomes, etc).
• sample_estimations.html Visualize and explore the samples, looking for outliers.
• differential_expression.html Perform differential expression analyses.
• gene_families.html Visualize specific gene families.
• gene_ontology.html Perform ontology analyses.
• gene_ontology_relaxed_p_input.html Perform ontology analyses with relaxed input.
• gene_ontology_relaxed_fc_input.html Perform ontology analyses with relaxed input.
• kegg.html KEGG analyses

This document is concerned with analyzing RNASeq data of human and parasites during infection. Two different cruzi strains were used: CL-14 and CL-Brener. I am not certain the infection conditions, unfortunately – I think (thank you, Edson!) that they are human foreskin fibroblasts.

2 TODO list

2.1 2016-11-16

1. Include with the significance bar plots the table describing the number of genes at each cutoff and put it to the right of the graph.
2. Redo significance bar plots with the following 3 contrasts: tryp to a60, a60 to a96, and a96 to tryp

2.2 2016-10-24

1. Different colored ma plots with gene families having fun colors.

2.3 2016-10-05

1. Bar plot of DE genes up/down as per figure 4 of the leishmania paper.

2.4 2016-09-25

1. Distance/Correlation heatmap(s) for sample estimation
   • Do these include CL14 epimastigote samples? If not, why?
   • Just in case, perform one with and one without.
2. PCA analysis ibid.
3. MA plots of limma DE contrasts:
   • epi/tryp CLBr
   • a60/tryp CLBr
   • a96/a60 CLBr
   • tryp/a96 CLBr
4. I think Santuza sent an email with further instructions.

2.4.1 The above from an email I just wrote

1. figures_atb/figure_02
   1. 1 svg image for each heatmap as I think you prefer (done)
   2. 1 powerpoint slide containing the same images laid out as per (done) the existing Fig2.pptx
2. tables_atb/table_s03
   1. 1 xlsx imitating that found in S3 Table.xlsx but including epi/tryp CLBr and lacking ncRNA features.
3. figures_atb/figure_03
   1. 1 svg image for each ma plot
   2. 1 powerpoint … as per 1b above.
4. figures_atb/figure_05
   1. ibid.
5. worksheets_atb/
   1. annotations.html: Actions performed to create a set of annotations for #2 lacking ncRNAs.
   2. sample_estimations.html: Actions performed for #1.
   3. differential_expression.html: Actions performed for #2,3,4.

2.5 Previous

1. SRA submission for CL14/CLBr samples
2. Read through methods and ensure mapping parameters etc make sense.

3 RNAsequencing of CL14 and CLBrener

This document attempts to perform analyses of an RNA sequencing experiment to compare the RNA expression profiles of the T. cruzi strains: CL14 and CLBrener. These collections were performed with Epimastigotes, Trypomastigotes, and Amastigotes at times 60 and 96 hours after infection.

4 Setting up

The following block may be used to set up required libraries and make sure everything is ready to run.

## First ensure that devtools is installed
devp <- try(library(devtools))
if (class(devp) == "try-error") {
    install.packages("devtools")
}

## Once that is installed, grab a fresh copy of my analysis code:
devtools::install_github("abelew/hpgltools")

pander::pander(sessionInfo())

R version 3.3.3 RC (2017-02-27 r72279)

**Platform:** x86_64-pc-linux-gnu (64-bit)

locale: LC_CTYPE=en_US.UTF-8, LC_NUMERIC=C, LC_TIME=en_US.UTF-8, LC_COLLATE=en_US.UTF-8, LC_MONETARY=en_US.UTF-8, LC_MESSAGES=en_US.UTF-8, LC_PAPER=en_US.UTF-8, LC_NAME=C, LC_ADDRESS=C, LC_TELEPHONE=C, LC_MEASUREMENT=en_US.UTF-8 and LC_IDENTIFICATION=C

attached base packages: stats, graphics, grDevices, utils, datasets and base

other attached packages: hpgltools(v.2017.01) and rmarkdown(v.1.3)

loaded via a namespace (and not attached): Rcpp(v.0.12.9), xml2(v.1.1.1), roxygen2(v.6.0.1), knitr(v.1.15.1), magrittr(v.1.5), BiocGenerics(v.0.20.0), devtools(v.1.12.0), munsell(v.0.4.3), colorspace(v.1.3-2), R6(v.2.2.0), foreach(v.1.4.3), stringr(v.1.2.0), plyr(v.1.8.4), tools(v.3.3.3), parallel(v.3.3.3), grid(v.3.3.3), Biobase(v.2.34.0), data.table(v.1.10.4), gtable(v.0.2.0), withr(v.1.0.2), commonmark(v.1.1), htmltools(v.0.3.5), iterators(v.1.0.8), yaml(v.2.1.14), lazyeval(v.0.2.0), rprojroot(v.1.2), digest(v.0.6.12), assertthat(v.0.1), tibble(v.1.2), crayon(v.1.3.2), ggplot2(v.2.2.1), base64enc(v.0.1-3), codetools(v.0.2-15), testthat(v.1.0.2), memoise(v.1.0.0), evaluate(v.0.10), stringi(v.1.1.2), pander(v.0.6.0), methods(v.3.3.3), scales(v.0.4.1) and backports(v.1.0.5)