Last updated: 2020-03-05

Checks: 7 0

Knit directory: 20170327_Psen2S4Ter_RNASeq/

This reproducible R Markdown analysis was created with workflowr (version 1.6.0). The Checks tab describes the reproducibility checks that were applied when the results were created. The Past versions tab lists the development history.


Great! Since the R Markdown file has been committed to the Git repository, you know the exact version of the code that produced these results.

Great job! The global environment was empty. Objects defined in the global environment can affect the analysis in your R Markdown file in unknown ways. For reproduciblity it’s best to always run the code in an empty environment.

The command set.seed(20200119) was run prior to running the code in the R Markdown file. Setting a seed ensures that any results that rely on randomness, e.g. subsampling or permutations, are reproducible.

Great job! Recording the operating system, R version, and package versions is critical for reproducibility.

Nice! There were no cached chunks for this analysis, so you can be confident that you successfully produced the results during this run.

Great job! Using relative paths to the files within your workflowr project makes it easier to run your code on other machines.

Great! You are using Git for version control. Tracking code development and connecting the code version to the results is critical for reproducibility. The version displayed above was the version of the Git repository at the time these results were generated.

Note that you need to be careful to ensure that all relevant files for the analysis have been committed to Git prior to generating the results (you can use wflow_publish or wflow_git_commit). workflowr only checks the R Markdown file, but you know if there are other scripts or data files that it depends on. Below is the status of the Git repository when the results were generated:


Ignored files:
    Ignored:    .Rhistory
    Ignored:    .Rproj.user/

Unstaged changes:
    Modified:   output/Enrichment_Hom_V_Het.csv
    Modified:   output/Enrichment_Mutant_V_WT.csv

Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.


These are the previous versions of the R Markdown and HTML files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view them.

File Version Author Date Message
Rmd f75f82a Steve Ped 2020-03-05 Added post-CQN PCA
html 86ffa07 Steve Ped 2020-02-20 Updated saample layout figure
Rmd 4d2af23 Steve Ped 2020-02-20 Modified heatmap sizes & initial network layout
html 876e40f Steve Ped 2020-02-17 Compiled after minor corrections
html 1032a6d Steve Ped 2020-01-28 Added link to HomVsHet
Rmd 9104ecd Steve Ped 2020-01-28 First draft of Hom Vs Het
html 468e6e3 Steve Ped 2020-01-28 Ran enrichment of Mut Vs WT
Rmd 3a9933c Steve Ped 2020-01-28 Finished Enrichment analysis on MutVsWt
html b086aff Steve Ped 2020-01-26 Added first step of enrichment analysis
Rmd ba189be Steve Ped 2020-01-26 Corrected packages
html 96d8cc7 Steve Ped 2020-01-25 Compiled after data export & added compression to output
Rmd 10a285b Steve Ped 2020-01-22 Expanded description in index and tried reusing an image
html 10a285b Steve Ped 2020-01-22 Expanded description in index and tried reusing an image
html b096ad9 Steve Ped 2020-01-21 Recompiled index
Rmd 01512da Steve Ped 2020-01-21 Added initial DE analysis to index
Rmd fbb6242 Steve Ped 2020-01-21 Paused DE analysis
html fbb6242 Steve Ped 2020-01-21 Paused DE analysis
html f154205 Steve Ped 2020-01-20 Got rid of pointless warning
Rmd bc12101 Steve Ped 2020-01-20 Added bash pipeline
html bc12101 Steve Ped 2020-01-20 Added bash pipeline
html f65bf66 Steve Ped 2020-01-20 Removed License & updated text for PCA
Rmd 39d0381 Steve Ped 2020-01-20 Finished QC
html 39d0381 Steve Ped 2020-01-20 Finished QC
Rmd acb4f8a Steve Ped 2020-01-19 Start workflowr project.

library(tidyverse)
library(tidygraph)
library(ggraph)
samples <- read_csv("data/samples.csv") %>%
    distinct(sampleName, .keep_all = TRUE) %>%
    dplyr::select(sample = sampleName, sampleID, genotype) %>%
    mutate(genotype = factor(genotype, levels = c("WT", "Het", "Hom")))

This dataset is an analysis of RNASeq data from a 3-way comparison of WT zebrafish with Heterozygous mutants (psen2S4Ter/+) and Homozygous mutants psen2S4Ter/S4Ter. The psen2S4Ter mutant allele is expected to be a premature termination of the psen2 gene, but instead an alternate downstream start site resulted in a near full-length transcript with significant similarity to a conventional Early Onset Familial Alzhemier’s Disease mutation.

The expected comparisons were intended to be a three-way group comparison, however the final model was defined by fitting the wild-type samples, differences between heterozygotes and wild-type, followed by the difference between the two mutant genotypes

create_ring(3) %>% 
  mutate(
    name = paste0(levels(samples$genotype), "\n(n = 4)"),
    name = factor(name, levels = name)
  ) %>% 
  activate(edges) %>%
  mutate(comparison = c("Het Vs WT", "Hom Vs Het", "Hom Vs WT")) %>% 
    dplyr::filter(comparison != "Hom Vs WT") %>%
  ggraph(layout = "kk") + 
  geom_edge_link2(
    aes(label = comparison),
    angle_calc = "along",
    label_dodge = unit(0.02, "npc"),
    start_cap = circle(0.09, "npc"),
    end_cap = circle(0.09, "npc"),
    label_size = 5,
    arrow = arrow(
      length = unit(0.06, "npc"),
      ends = "both", 
      type = "closed"
    )
  ) +
  geom_node_label(
    aes(label = name, colour = name),
    size = 5,
    fill = rgb(1,1,1,0.7),
    label.padding = unit(0.4, "lines")
  ) + 
  scale_y_continuous(expand = expand_scale(0.1)) +
  scale_x_continuous(expand = expand_scale(0.1)) +
  theme_void() +
  theme(
    legend.position = "none"
  ) 

Version Author Date
86ffa07 Steve Ped 2020-02-20
10a285b Steve Ped 2020-01-22

All samples were female, 6 months old and raised in the same tank as a family, to minimise variability. This age represents sexual maturity and is expected to model a pre-symptomatic brain. Fish were all killed and genotyped by tail-clipping on 07-07-2016.

RNAseq was performed on total RNA (i.e. ribo-reduced) from whole brain tissue, with \(n=4\) samples in each group. Sequencing was performed by the sequencing facility at the Centre for Cancer Biology in Adelaide, using an Illimuna NextSeq. Reads were provided in as paired-end, 150bp reads.

  1. Bash Pipeline
  2. Quality Assesment
  3. Differential Expression Analysis
  4. Enrichment Analysis
    1. Mutant Vs Wild Type
    2. Homozygous Vs Heterzygous Mutants

devtools::session_info()
─ Session info ───────────────────────────────────────────────────────────────
 setting  value                       
 version  R version 3.6.3 (2020-02-29)
 os       Ubuntu 18.04.4 LTS          
 system   x86_64, linux-gnu           
 ui       X11                         
 language en_AU:en                    
 collate  en_AU.UTF-8                 
 ctype    en_AU.UTF-8                 
 tz       Australia/Melbourne         
 date     2020-03-05                  

─ Packages ───────────────────────────────────────────────────────────────────
 package      * version  date       lib source        
 assertthat     0.2.1    2019-03-21 [2] CRAN (R 3.6.0)
 backports      1.1.5    2019-10-02 [2] CRAN (R 3.6.1)
 broom          0.5.4    2020-01-27 [2] CRAN (R 3.6.2)
 callr          3.4.2    2020-02-12 [2] CRAN (R 3.6.2)
 cellranger     1.1.0    2016-07-27 [2] CRAN (R 3.6.0)
 cli            2.0.1    2020-01-08 [2] CRAN (R 3.6.2)
 colorspace     1.4-1    2019-03-18 [2] CRAN (R 3.6.0)
 crayon         1.3.4    2017-09-16 [2] CRAN (R 3.6.0)
 DBI            1.1.0    2019-12-15 [2] CRAN (R 3.6.2)
 dbplyr         1.4.2    2019-06-17 [2] CRAN (R 3.6.0)
 desc           1.2.0    2018-05-01 [2] CRAN (R 3.6.0)
 devtools       2.2.2    2020-02-17 [2] CRAN (R 3.6.2)
 digest         0.6.25   2020-02-23 [2] CRAN (R 3.6.2)
 dplyr        * 0.8.4    2020-01-31 [2] CRAN (R 3.6.2)
 ellipsis       0.3.0    2019-09-20 [2] CRAN (R 3.6.1)
 evaluate       0.14     2019-05-28 [2] CRAN (R 3.6.0)
 fansi          0.4.1    2020-01-08 [2] CRAN (R 3.6.2)
 farver         2.0.3    2020-01-16 [2] CRAN (R 3.6.2)
 forcats      * 0.4.0    2019-02-17 [2] CRAN (R 3.6.0)
 fs             1.3.1    2019-05-06 [2] CRAN (R 3.6.0)
 generics       0.0.2    2018-11-29 [2] CRAN (R 3.6.0)
 ggforce        0.3.1    2019-08-20 [2] CRAN (R 3.6.1)
 ggplot2      * 3.2.1    2019-08-10 [2] CRAN (R 3.6.1)
 ggraph       * 2.0.1    2020-02-07 [2] CRAN (R 3.6.2)
 ggrepel        0.8.1    2019-05-07 [2] CRAN (R 3.6.0)
 git2r          0.26.1   2019-06-29 [2] CRAN (R 3.6.1)
 glue           1.3.1    2019-03-12 [2] CRAN (R 3.6.0)
 graphlayouts   0.5.0    2019-08-20 [2] CRAN (R 3.6.1)
 gridExtra      2.3      2017-09-09 [2] CRAN (R 3.6.0)
 gtable         0.3.0    2019-03-25 [2] CRAN (R 3.6.0)
 haven          2.2.0    2019-11-08 [2] CRAN (R 3.6.1)
 hms            0.5.3    2020-01-08 [2] CRAN (R 3.6.2)
 htmltools      0.4.0    2019-10-04 [2] CRAN (R 3.6.1)
 httpuv         1.5.2    2019-09-11 [2] CRAN (R 3.6.1)
 httr           1.4.1    2019-08-05 [2] CRAN (R 3.6.1)
 igraph         1.2.4.2  2019-11-27 [2] CRAN (R 3.6.1)
 jsonlite       1.6.1    2020-02-02 [2] CRAN (R 3.6.2)
 knitr          1.28     2020-02-06 [2] CRAN (R 3.6.2)
 labeling       0.3      2014-08-23 [2] CRAN (R 3.6.0)
 later          1.0.0    2019-10-04 [2] CRAN (R 3.6.1)
 lattice        0.20-40  2020-02-19 [4] CRAN (R 3.6.2)
 lazyeval       0.2.2    2019-03-15 [2] CRAN (R 3.6.0)
 lifecycle      0.1.0    2019-08-01 [2] CRAN (R 3.6.1)
 lubridate      1.7.4    2018-04-11 [2] CRAN (R 3.6.0)
 magrittr       1.5      2014-11-22 [2] CRAN (R 3.6.0)
 MASS           7.3-51.5 2019-12-20 [4] CRAN (R 3.6.2)
 memoise        1.1.0    2017-04-21 [2] CRAN (R 3.6.0)
 modelr         0.1.6    2020-02-22 [2] CRAN (R 3.6.2)
 munsell        0.5.0    2018-06-12 [2] CRAN (R 3.6.0)
 nlme           3.1-144  2020-02-06 [4] CRAN (R 3.6.2)
 pillar         1.4.3    2019-12-20 [2] CRAN (R 3.6.2)
 pkgbuild       1.0.6    2019-10-09 [2] CRAN (R 3.6.1)
 pkgconfig      2.0.3    2019-09-22 [2] CRAN (R 3.6.1)
 pkgload        1.0.2    2018-10-29 [2] CRAN (R 3.6.0)
 polyclip       1.10-0   2019-03-14 [2] CRAN (R 3.6.0)
 prettyunits    1.1.1    2020-01-24 [2] CRAN (R 3.6.2)
 processx       3.4.2    2020-02-09 [2] CRAN (R 3.6.2)
 promises       1.1.0    2019-10-04 [2] CRAN (R 3.6.1)
 ps             1.3.2    2020-02-13 [2] CRAN (R 3.6.2)
 purrr        * 0.3.3    2019-10-18 [2] CRAN (R 3.6.1)
 R6             2.4.1    2019-11-12 [2] CRAN (R 3.6.1)
 Rcpp           1.0.3    2019-11-08 [2] CRAN (R 3.6.1)
 readr        * 1.3.1    2018-12-21 [2] CRAN (R 3.6.0)
 readxl         1.3.1    2019-03-13 [2] CRAN (R 3.6.0)
 remotes        2.1.1    2020-02-15 [2] CRAN (R 3.6.2)
 reprex         0.3.0    2019-05-16 [2] CRAN (R 3.6.0)
 rlang          0.4.4    2020-01-28 [2] CRAN (R 3.6.2)
 rmarkdown      2.1      2020-01-20 [2] CRAN (R 3.6.2)
 rprojroot      1.3-2    2018-01-03 [2] CRAN (R 3.6.0)
 rstudioapi     0.11     2020-02-07 [2] CRAN (R 3.6.2)
 rvest          0.3.5    2019-11-08 [2] CRAN (R 3.6.1)
 scales         1.1.0    2019-11-18 [2] CRAN (R 3.6.1)
 sessioninfo    1.1.1    2018-11-05 [2] CRAN (R 3.6.0)
 stringi        1.4.6    2020-02-17 [2] CRAN (R 3.6.2)
 stringr      * 1.4.0    2019-02-10 [2] CRAN (R 3.6.0)
 testthat       2.3.1    2019-12-01 [2] CRAN (R 3.6.1)
 tibble       * 2.1.3    2019-06-06 [2] CRAN (R 3.6.0)
 tidygraph    * 1.1.2    2019-02-18 [2] CRAN (R 3.6.0)
 tidyr        * 1.0.2    2020-01-24 [2] CRAN (R 3.6.2)
 tidyselect     1.0.0    2020-01-27 [2] CRAN (R 3.6.2)
 tidyverse    * 1.3.0    2019-11-21 [2] CRAN (R 3.6.1)
 tweenr         1.0.1    2018-12-14 [2] CRAN (R 3.6.0)
 usethis        1.5.1    2019-07-04 [2] CRAN (R 3.6.1)
 vctrs          0.2.3    2020-02-20 [2] CRAN (R 3.6.2)
 viridis        0.5.1    2018-03-29 [2] CRAN (R 3.6.0)
 viridisLite    0.3.0    2018-02-01 [2] CRAN (R 3.6.0)
 whisker        0.4      2019-08-28 [2] CRAN (R 3.6.1)
 withr          2.1.2    2018-03-15 [2] CRAN (R 3.6.0)
 workflowr    * 1.6.0    2019-12-19 [2] CRAN (R 3.6.2)
 xfun           0.12     2020-01-13 [2] CRAN (R 3.6.2)
 xml2           1.2.2    2019-08-09 [2] CRAN (R 3.6.1)
 yaml           2.2.1    2020-02-01 [2] CRAN (R 3.6.2)

[1] /home/steveped/R/x86_64-pc-linux-gnu-library/3.6
[2] /usr/local/lib/R/site-library
[3] /usr/lib/R/site-library
[4] /usr/lib/R/library