From 9d53bdf81675d899b628ab9a19271a1958aae1a5 Mon Sep 17 00:00:00 2001 From: Chris Stubben Date: Tue, 24 Aug 2021 16:23:09 -0600 Subject: [PATCH] update vignettes --- vignettes/Pasilla.Rmd | 50 +++++------ vignettes/Pasilla.md | 80 +++++++++--------- .../Pasilla_files/figure-gfm/pcaplot-1.png | Bin 28830 -> 28496 bytes .../Pasilla_files/figure-gfm/plot1-1.png | Bin 67145 -> 67147 bytes .../Pasilla_files/figure-gfm/volcano-1.png | Bin 64980 -> 65504 bytes 5 files changed, 67 insertions(+), 63 deletions(-) diff --git a/vignettes/Pasilla.Rmd b/vignettes/Pasilla.Rmd index 799fbf1..31e1015 100644 --- a/vignettes/Pasilla.Rmd +++ b/vignettes/Pasilla.Rmd @@ -9,7 +9,7 @@ vignette: > --- ```{r global_options, include=FALSE} -knitr::opts_chunk$set(warning=FALSE, comment="# ", collapse=TRUE) +knitr::opts_chunk$set(comment="# ", collapse=TRUE) ``` This guide follows the [Bioconductor RNA-Seq workflow] to find differentially @@ -62,35 +62,35 @@ counts <- filter_counts(counts, n = 5) ### Load annotations The [hciRdata] package includes the latest Ensembl annotations for -twelve common reference genomes (version 98). +twelve common reference genomes. -```{r fly98, echo=-1} +```{r fly104, echo=-1} options(width=110) library(hciRdata) -dplyr::select(fly98, 1:4,8) +# data(package="hciRdata") +dplyr::select(fly104, 1:4,8) ``` -The [pasilla] dataset was analyzed using Ensembl version 62, so 708 gene ids have -been removed from the latest release. See the [Ensembl] vignette to download an + +The [pasilla] dataset was analyzed using Ensembl version 62, so 737 gene ids have +been removed from the latest release. See the [Ensembl] vignette to download and use an earlier release. ```{r missinggene} -filter(counts, !id %in% fly98$id) %>% nrow() +filter(counts, !id %in% fly104$id) %>% nrow() ``` - Check genes with the highest number of assigned reads. ```{r topgenes, echo=-1} options(width=110) n1 <- rowMeans(as_matrix(counts)) -right_join( dplyr::select(fly98, 1:4,8), +right_join( dplyr::select(fly104, 1:4,8), tibble(id= names(n1), mean_count = n1)) %>% arrange(desc(mean_count)) ``` - ### Run DESeq Run `DESeq` using ~ condition + type in the design formula to control for @@ -130,31 +130,31 @@ plot_dist(rld , c("condition", "type"), na_col="white") ### Results +DESeq will convert any character columns in the design formula to factors (see +warning above). In this case, the values will be ordered alphabetically and the +`results_all` function will run ***all*** pairwise comparisons in reverse order +(for example C vs. B, C vs. A and B vs. A if samples groups are A, B, C). -The `results_all` function will run ***all*** pairwise comparisons -in alphabetical order (A vs B, A vs C and B vs C). Use `check_contrasts` to -list the contrasts. +Check the possible contrasts using `check_contrasts`. - -```{r checkcontrasts} +```{r check_condition} check_contrasts(dds$condition) ``` -The control group should be the reference level and listed second. If you need to reorder the -groups, use `factor` to set the factor levels ***before*** running `DESeq` above. For example, -if you have a `trt` column with KO and control groups, set the factor levels so the control group -is listed last. Note the standard DESeq2 workflow typically wants the control group listed first. +The best way to ensure that control groups are set as the reference level is to factor +the columns before running `deseq_from_tibble`. -```{r relevel, eval=FALSE} -samples$trt <- factor(samples$trt, levels = c( "KO", "control")) -``` -Compare treated vs. untreated using a 5% FDR and add gene annotations to the result tables. +```{r factor_condition, eval=FALSE} +samples$condition <- factor(samples$condition, levels = c("untreated", "treated")) +``` +Another option is to use the relevel option in `results_all` below to compare +treated vs. untreated using a 5% FDR (or setting `vs="untreated"` would also work in this case). ```{r DESeq_results} -res <- results_all(dds, fly98, alpha= 0.05) +res <- results_all(dds, fly104, alpha= 0.05, relevel =c("untreated", "treated")) ``` The results are a tibble or list of tibbles if there is more than one contrast. @@ -203,7 +203,7 @@ and fly annotations to a single Excel file in `DESeq.xlsx` and R objects to a binary data file to load into a new session. ```{r write_results_to_Excel, eval=FALSE} -write_deseq(res, dds, rld, fly98) +write_deseq(res, dds, rld, fly104) save(res, dds, rld, file="dds.rda") ``` diff --git a/vignettes/Pasilla.md b/vignettes/Pasilla.md index 86e28b6..f151d75 100644 --- a/vignettes/Pasilla.md +++ b/vignettes/Pasilla.md @@ -1,12 +1,12 @@ DESeq analysis of Pasilla knock-downs ================ -October 18, 2019 +August 24, 2021 This guide follows the [Bioconductor RNA-Seq workflow](http://master.bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html) to find differentially expressed genes using [DESeq2](http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) -version 1.24.0. The +version 1.30.1. The [hciR](https://github.com/HuntsmanCancerInstitute/hciR) package works best with [tidyverse](http://tidyverse.org/) packages (readr, dplyr, tibble, etc.) and simplifies the code in a typical differential @@ -83,12 +83,13 @@ counts <- filter_counts(counts, n = 5) The [hciRdata](https://github.com/HuntsmanCancerInstitute/hciRdata) package includes the latest Ensembl annotations for twelve common -reference genomes (version 98). +reference genomes. ``` r library(hciRdata) -dplyr::select(fly98, 1:4,8) -# # A tibble: 17,753 x 5 +# data(package="hciRdata") +dplyr::select(fly104, 1:4,8) +# # A tibble: 23,932 x 5 # id gene_name biotype chromosome description # # 1 FBgn0000003 7SLRNA:CR32864 ncRNA 3R Signal recognition particle 7SL RNA CR32864 @@ -101,26 +102,26 @@ dplyr::select(fly98, 1:4,8) # 8 FBgn0000024 Ace protein_coding 3R Acetylcholine esterase # 9 FBgn0000028 acj6 protein_coding X abnormal chemosensory jump 6 # 10 FBgn0000032 Acph-1 protein_coding 3R Acid phosphatase 1 -# # … with 17,743 more rows +# # … with 23,922 more rows ``` The [pasilla](http://bioconductor.org/packages/release/data/experiment/html/pasilla.html) -dataset was analyzed using Ensembl version 62, so 708 gene ids have been +dataset was analyzed using Ensembl version 62, so 737 gene ids have been removed from the latest release. See the [Ensembl](https://github.com/HuntsmanCancerInstitute/hciR/vignettes/Ensembl.md) -vignette to download an earlier release. +vignette to download and use an earlier release. ``` r -filter(counts, !id %in% fly98$id) %>% nrow() -# [1] 708 +filter(counts, !id %in% fly104$id) %>% nrow() +# [1] 737 ``` Check genes with the highest number of assigned reads. ``` r n1 <- rowMeans(as_matrix(counts)) -right_join( dplyr::select(fly98, 1:4,8), +right_join( dplyr::select(fly104, 1:4,8), tibble(id= names(n1), mean_count = n1)) %>% arrange(desc(mean_count)) # Joining, by = "id" @@ -142,7 +143,7 @@ right_join( dplyr::select(fly98, 1:4,8), ### Run DESeq -Run `DESeq` using ~ condition + type in the design formula to control +Run `DESeq` using \~ condition + type in the design formula to control for paired vs single end effects on gene expression and get the regularized log (rlog) counts for sample visualizations. These values are similar to the log2 normalized counts except the variance in low @@ -151,6 +152,8 @@ count genes is reduced. ``` r dds <- deseq_from_tibble(counts, samples, design = ~ condition + type) # Reordering columns in counts to match samples +# Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in design formula are characters, +# converting to factors # estimating size factors # estimating dispersions # gene-wise dispersion estimates @@ -187,36 +190,37 @@ plot_dist(rld , c("condition", "type"), na_col="white") ### Results -The `results_all` function will run ***all*** pairwise comparisons in -alphabetical order (A vs B, A vs C and B vs C). Use `check_contrasts` to -list the contrasts. +DESeq will convert any character columns in the design formula to +factors (see warning above). In this case, the values will be ordered +alphabetically and the `results_all` function will run ***all*** +pairwise comparisons in reverse order (for example C vs. B, C vs. A and +B vs. A if samples groups are A, B, C). + +Check the possible contrasts using `check_contrasts`. ``` r check_contrasts(dds$condition) # 1 contrasts: -# [1] "treated vs. untreated" +# [1] "untreated vs. treated" ``` -The control group should be the reference level and listed second. If -you need to reorder the groups, use `factor` to set the factor levels -***before*** running `DESeq` above. For example, if you have a `trt` -column with KO and control groups, set the factor levels so the control -group is listed last. Note the standard DESeq2 workflow typically wants -the control group listed first. +The best way to ensure that control groups are set as the reference +level is to factor the columns before running `deseq_from_tibble`. ``` r -samples$trt <- factor(samples$trt, levels = c( "KO", "control")) +samples$condition <- factor(samples$condition, levels = c("untreated", "treated")) ``` -Compare treated vs. untreated using a 5% FDR and add gene annotations to -the result tables. +Another option is to use the relevel option in `results_all` below to +compare treated vs. untreated using a 5% FDR (or setting +`vs="untreated"` would also work in this case). ``` r -res <- results_all(dds, fly98, alpha= 0.05) +res <- results_all(dds, fly104, alpha= 0.05, relevel =c("untreated", "treated")) # Using adjusted p-value < 0.05 # Adding shrunken fold changes to log2FoldChange # 1. treated vs. untreated: 492 up and 598 down regulated -# Note: 708 rows in results are missing from biomart table +# Note: 737 rows in results are missing from biomart table ``` The results are a tibble or list of tibbles if there is more than one @@ -228,16 +232,16 @@ arrange(res, padj) %>% # # A tibble: 9,405 x 8 # id gene_name biotype chromosome description baseMean log2FoldChange padj # -# 1 FBgn0003… sesB protein_cod… X stress-sensitive B 4342. -2.96 2.13e-176 -# 2 FBgn0026… SPARC protein_cod… 3R Secreted protein, acidic, cy… 43914. -2.38 4.34e-170 -# 3 FBgn0039… Kal1 protein_cod… 3R Kallmann syndrome 1 730. -4.09 3.74e-166 -# 4 FBgn0025… Ant2 protein_cod… X Adenine nucleotide transloca… 1501. 2.71 3.06e-160 -# 5 FBgn0029… Hml protein_cod… 3L Hemolectin 3706. -2.10 5.31e-111 -# 6 FBgn0035… CG3770 protein_cod… 2R 638. -2.39 9.53e- 86 -# 7 FBgn0039… CG1544 protein_cod… 3R 262. -3.46 2.15e- 80 -# 8 FBgn0034… gas protein_cod… 2R gasoline 226. -2.96 9.24e- 64 -# 9 FBgn0000… Ama protein_cod… 3R Amalgam 342. 2.36 7.82e- 59 -# 10 FBgn0029… CG3168 protein_cod… X 490. -2.22 8.59e- 59 +# 1 FBgn0003… sesB protein_cod… X "stress-sensitive B" 4342. -2.96 2.13e-176 +# 2 FBgn0026… SPARC protein_cod… 3R "Secreted protein, acidic, c… 43914. -2.38 4.34e-170 +# 3 FBgn0039… Kal1 protein_cod… 3R "Kallmann syndrome 1" 730. -4.09 3.74e-166 +# 4 FBgn0025… Ant2 protein_cod… X "Adenine nucleotide transloc… 1501. 2.71 3.06e-160 +# 5 FBgn0029… Hml protein_cod… 3L "Hemolectin" 3706. -2.10 5.31e-111 +# 6 FBgn0035… CG3770 protein_cod… 2R "" 638. -2.39 9.53e- 86 +# 7 FBgn0039… CG1544 protein_cod… 3R "" 262. -3.46 2.15e- 80 +# 8 FBgn0034… gas protein_cod… 2R "gasoline" 226. -2.96 9.24e- 64 +# 9 FBgn0000… Ama protein_cod… 3R "Amalgam" 342. 2.36 7.82e- 59 +# 10 FBgn0029… CG3168 protein_cod… X "" 490. -2.22 8.59e- 59 # # … with 9,395 more rows ``` @@ -293,7 +297,7 @@ counts and fly annotations to a single Excel file in `DESeq.xlsx` and R objects to a binary data file to load into a new session. ``` r -write_deseq(res, dds, rld, fly98) +write_deseq(res, dds, rld, fly104) save(res, dds, rld, file="dds.rda") ``` 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