Normalization and Variance Modelling
21 July 2023
library(scater)
library(scran)
library(BiocStyle)
library(pheatmap)
sce.paul <- readRDS("output/paul_quality_control_sce.RDS")Normalization
library(scran)
set.seed(101000110)
clusters <- quickCluster(sce.paul)
sce.paul <- computeSumFactors(sce.paul, clusters=clusters)
sce.paul <- logNormCounts(sce.paul)We examine some key metrics for the distribution of size factors, and compare it to the library sizes as a sanity check.
summary(sizeFactors(sce.paul)) Min. 1st Qu. Median Mean 3rd Qu. Max.
0.05681 0.42248 0.77545 1.00000 1.33514 9.65387 plot(librarySizeFactors(sce.paul), sizeFactors(sce.paul), pch=16,
xlab="Library size factors", ylab="Deconvolution factors", log="xy")
Relationship between the library size factors and the deconvolution size factors in the Paul HSC dataset.
Variance modelling
We fit a mean-variance trend to the endogenous genes to detect highly variable genes.
Unfortunately, the plates are confounded with an experimental treatment (Batch_desc) so we cannot block on the plate of origin.
set.seed(00010101)
dec.paul <- modelGeneVarByPoisson(sce.paul)
top.paul <- getTopHVGs(dec.paul, prop=0.1)plot(dec.paul$mean, dec.paul$total, pch=16, cex=0.5,
xlab="Mean of log-expression", ylab="Variance of log-expression")
curve(metadata(dec.paul)$trend(x), col="blue", add=TRUE)
Per-gene variance as a function of the mean for the log-expression values in the Paul HSC dataset. Each point represents a gene (black) with the mean-variance trend (blue) fitted to simulated Poisson noise.
saveRDS(sce.paul,"output/paul_normalization_sce.RDS")
saveRDS(dec.paul,"output/paul_normalization_dec.RDS")
saveRDS(top.paul,"output/paul_normalization_top.RDS")Click to see page metadata
Computation Started: 2023-07-21 16:24:08
Finished in 1.003 mins
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Packages
| package | version | date |
|---|---|---|
| MatrixGenerics | 1.2.0 | 2020-10-28 |
| Biobase | 2.50.0 | 2020-10-28 |
| viridis | 0.5.1 | 2020-07-17 |
| edgeR | 3.32.1 | 2021-01-15 |
| BiocSingular | 1.6.0 | 2020-10-28 |
| viridisLite | 0.3.0 | 2020-06-15 |
| DelayedMatrixStats | 1.12.2 | 2021-01-13 |
| scuttle | 1.0.4 | 2020-12-18 |
| assertthat | 0.2.1 | 2020-07-15 |
| statmod | 1.4.35 | 2020-10-20 |
| highr | 0.8 | 2020-07-15 |
| BiocManager | 1.30.10 | 2020-07-15 |
| stats4 | 4.0.1 | 2020-06-07 |
| dqrng | 0.2.1 | 2020-07-15 |
| grDevices | 4.0.1 | 2020-06-07 |
| GenomeInfoDbData | 1.2.4 | 2020-11-03 |
| vipor | 0.4.5 | 2020-07-15 |
| yaml | 2.2.1 | 2020-07-15 |
| pillar | 1.6.0 | 2021-04-14 |
| lattice | 0.20-41 | 2020-06-07 |
| glue | 1.4.2 | 2020-08-28 |
| base | 4.0.1 | 2020-06-07 |
| limma | 3.46.0 | 2020-10-28 |
| beachmat | 2.6.4 | 2020-12-21 |
| digest | 0.6.27 | 2020-10-25 |
| RColorBrewer | 1.1-2 | 2020-07-15 |
| GenomicRanges | 1.42.0 | 2020-10-28 |
| XVector | 0.30.0 | 2020-10-29 |
| colorspace | 2.0-0 | 2020-11-12 |
| htmltools | 0.5.5 | 2023-07-17 |
| Matrix | 1.2-18 | 2020-06-07 |
| pkgconfig | 2.0.3 | 2020-07-15 |
| pheatmap | 1.0.12 | 2020-07-16 |
| zlibbioc | 1.36.0 | 2020-10-29 |
| purrr | 0.3.4 | 2020-07-15 |
| scales | 1.1.1 | 2020-07-16 |
| BiocParallel | 1.24.1 | 2020-11-07 |
| git2r | 0.28.0 | 2021-01-11 |
| tibble | 3.1.1 | 2021-04-19 |
| generics | 0.1.0 | 2020-11-01 |
| datasets | 4.0.1 | 2020-06-07 |
| IRanges | 2.24.1 | 2020-12-13 |
| ggplot2 | 3.3.3 | 2020-12-31 |
| ellipsis | 0.3.1 | 2020-07-15 |
| withr | 2.4.2 | 2021-04-19 |
| SummarizedExperiment | 1.20.0 | 2020-10-28 |
| BiocGenerics | 0.36.0 | 2020-10-28 |
| cli | 2.5.0 | 2021-04-27 |
| magrittr | 2.0.1 | 2020-11-18 |
| crayon | 1.4.1 | 2021-02-09 |
| evaluate | 0.14 | 2020-06-15 |
| methods | 4.0.1 | 2020-06-07 |
| fansi | 0.4.2 | 2021-01-16 |
| bluster | 1.0.0 | 2020-10-28 |
| utils | 4.0.1 | 2020-06-07 |
| beeswarm | 0.2.3 | 2020-07-15 |
| tools | 4.0.1 | 2020-06-07 |
| scater | 1.18.3 | 2020-11-09 |
| BiocStyle | 2.18.1 | 2020-11-25 |
| lifecycle | 1.0.0 | 2021-02-16 |
| matrixStats | 0.57.0 | 2020-09-26 |
| stringr | 1.4.0 | 2020-07-15 |
| S4Vectors | 0.28.1 | 2020-12-10 |
| munsell | 0.5.0 | 2020-07-15 |
| locfit | 1.5-9.4 | 2020-07-15 |
| DelayedArray | 0.16.0 | 2020-10-28 |
| irlba | 2.3.3 | 2020-07-15 |
| stats | 4.0.1 | 2020-06-07 |
| compiler | 4.0.1 | 2020-06-07 |
| GenomeInfoDb | 1.26.2 | 2020-12-09 |
| rsvd | 1.0.3 | 2020-07-15 |
| rlang | 1.1.1 | 2023-07-17 |
| grid | 4.0.1 | 2020-06-07 |
| RCurl | 1.98-1.2 | 2020-07-15 |
| BiocNeighbors | 1.8.2 | 2020-12-08 |
| graphics | 4.0.1 | 2020-06-07 |
| SingleCellExperiment | 1.12.0 | 2020-10-28 |
| igraph | 1.2.6 | 2020-10-07 |
| bitops | 1.0-6 | 2020-07-15 |
| rmarkdown | 2.23 | 2023-07-17 |
| gtable | 0.3.0 | 2020-07-15 |
| DBI | 1.1.1 | 2021-01-16 |
| R6 | 2.5.0 | 2020-10-29 |
| gridExtra | 2.3 | 2020-07-15 |
| knitr | 1.30 | 2020-09-23 |
| dplyr | 1.0.5 | 2021-03-06 |
| fastmap | 1.1.1 | 2023-07-17 |
| utf8 | 1.1.4 | 2020-07-15 |
| stringi | 1.5.3 | 2020-09-10 |
| ggbeeswarm | 0.6.0 | 2020-07-16 |
| parallel | 4.0.1 | 2020-06-07 |
| Rcpp | 1.0.6 | 2021-01-16 |
| scran | 1.18.3 | 2020-12-22 |
| vctrs | 0.3.6 | 2020-12-18 |
| tidyselect | 1.1.0 | 2020-07-15 |
| xfun | 0.39 | 2023-07-17 |
| sparseMatrixStats | 1.2.0 | 2020-10-28 |
System Information
| systemInfo | |
|---|---|
| version | R version 4.0.1 (2020-06-06) |
| platform | x86_64-apple-darwin17.0 (64-bit) |
| locale | en_CA.UTF-8 |
| OS | macOS 10.16 |
| UI | X11 |
Scikick Configuration
cat scikick.yml### Scikick Project Workflow Configuration File
# Directory where Scikick will store all standard notebook outputs
reportdir: report
# --- Content below here is best modified by using the Scikick CLI ---
# Notebook Execution Configuration (format summarized below)
# analysis:
# first_notebook.Rmd:
# second_notebook.Rmd:
# - first_notebook.Rmd # must execute before second_notebook.Rmd
# - functions.R # file is used by second_notebook.Rmd
#
# Each analysis item is executed to generate md and html files, E.g.:
# 1. <reportdir>/out_md/first_notebook.md
# 2. <reportdir>/out_html/first_notebook.html
analysis: !!omap
- index.Rmd:
- notebooks/nestorowa/import.Rmd:
- notebooks/nestorowa/quality_control.Rmd:
- notebooks/nestorowa/import.Rmd
- notebooks/nestorowa/normalization.Rmd:
- notebooks/nestorowa/quality_control.Rmd
- notebooks/nestorowa/further_exploration.Rmd:
- notebooks/nestorowa/normalization.Rmd
- notebooks/grun/import.Rmd:
- notebooks/grun/quality_control.Rmd:
- notebooks/grun/import.Rmd
- notebooks/grun/normalization.Rmd:
- notebooks/grun/quality_control.Rmd
- notebooks/grun/further_exploration.Rmd:
- notebooks/grun/normalization.Rmd
- notebooks/paul/import.Rmd:
- notebooks/paul/quality_control.Rmd:
- notebooks/paul/import.Rmd
- notebooks/paul/normalization.Rmd:
- notebooks/paul/quality_control.Rmd
- notebooks/paul/further_exploration.Rmd:
- notebooks/paul/normalization.Rmd
- notebooks/merged/merge.Rmd:
- notebooks/grun/quality_control.Rmd
- notebooks/paul/quality_control.Rmd
- notebooks/nestorowa/normalization.Rmd
- notebooks/merged/combined_analysis.Rmd:
- notebooks/merged/merge.Rmd
version_info:
snakemake: 6.0.2
ruamel.yaml: 0.16.12
scikick: 0.2.1
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