Last updated: 2024-07-29
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| Rmd | 0c10121 | Dave Tang | 2024-07-29 | Normalisation methods using edgeR |
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| Rmd | 4bc5f6a | Dave Tang | 2023-10-13 | edgeR normalisation |
Differential expression analysis of RNA-seq expression profiles with biological replication. Implements a range of statistical methodology based on the negative binomial distributions, including empirical Bayes estimation, exact tests, generalized linear models and quasi-likelihood tests. As well as RNA-seq, it be applied to differential signal analysis of other types of genomic data that produce read counts, including ChIP-seq, ATAC-seq, Bisulfite-seq, SAGE and CAGE.
Install using BiocManager::install().
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("edgeR")From ?edgeR::calcNormFactors:
Calculate scaling factors to convert the raw library sizes for a set of sequenced samples into normalized effective library sizes.
This function computes scaling factors to convert observed library sizes into normalized library sizes, also called “effective library sizes”. The effective library sizes for use in downstream analysis are lib.size * norm.factors where lib.size contains the original library sizes and norm.factors is the vector of scaling factors computed by this function.
The TMM method implements the trimmed mean of M-values method proposed by Robinson and Oshlack (2010). By default, the M-values are weighted according to inverse variances, as computed by the delta method for logarithms of binomial random variables. If refColumn is unspecified, then the column whose count-per-million upper quartile is closest to the mean upper quartile is set as the reference library.
The TMMwsp method stands for “TMM with singleton pairing”. This is a variant of TMM that is intended to have more stable performance when the counts have a high proportion of zeros. In the TMM method, genes that have zero count in either library are ignored when comparing pairs of libraries. In the TMMwsp method, the positive counts from such genes are reused to increase the number of features by which the libraries are compared. The singleton positive counts are paired up between the libraries in decreasing order of size and then a slightly modified TMM method is applied to the re-ordered libraries. If refColumn is unspecified, then the column with largest sum of square-root counts is used as the reference library.
RLE is the scaling factor method proposed by Anders and Huber (2010). We call it “relative log expression”, as median library is calculated from the geometric mean of all columns and the median ratio of each sample to the median library is taken as the scale factor.
The upperquartile method is the upper-quartile normalization method of Bullard et al (2010), in which the scale factors are calculated from the 75% quantile of the counts for each library, after removing genes that are zero in all libraries. The idea is generalized here to allow normalization by any quantile of the count distributions.
If method=“none”, then the normalization factors are set to 1.
For symmetry, normalization factors are adjusted to multiply to 1. Rows of object that have zero counts for all columns are removed before normalization factors are computed. The number of such rows does not affect the estimated normalization factors.
sessionInfo()R version 4.4.0 (2024-04-24)
Platform: x86_64-pc-linux-gnu
Running under: Ubuntu 22.04.4 LTS
Matrix products: default
BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.20.so; LAPACK version 3.10.0
locale:
[1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
time zone: Etc/UTC
tzcode source: system (glibc)
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] lubridate_1.9.3 forcats_1.0.0 stringr_1.5.1 dplyr_1.1.4
[5] purrr_1.0.2 readr_2.1.5 tidyr_1.3.1 tibble_3.2.1
[9] ggplot2_3.5.1 tidyverse_2.0.0 workflowr_1.7.1
loaded via a namespace (and not attached):
[1] sass_0.4.9 utf8_1.2.4 generics_0.1.3 stringi_1.8.4
[5] hms_1.1.3 digest_0.6.35 magrittr_2.0.3 timechange_0.3.0
[9] evaluate_0.24.0 grid_4.4.0 fastmap_1.2.0 rprojroot_2.0.4
[13] jsonlite_1.8.8 processx_3.8.4 whisker_0.4.1 ps_1.7.6
[17] promises_1.3.0 httr_1.4.7 fansi_1.0.6 scales_1.3.0
[21] jquerylib_0.1.4 cli_3.6.2 rlang_1.1.4 munsell_0.5.1
[25] withr_3.0.0 cachem_1.1.0 yaml_2.3.8 tools_4.4.0
[29] tzdb_0.4.0 colorspace_2.1-0 httpuv_1.6.15 vctrs_0.6.5
[33] R6_2.5.1 lifecycle_1.0.4 git2r_0.33.0 fs_1.6.4
[37] pkgconfig_2.0.3 callr_3.7.6 pillar_1.9.0 bslib_0.7.0
[41] later_1.3.2 gtable_0.3.5 glue_1.7.0 Rcpp_1.0.12
[45] xfun_0.44 tidyselect_1.2.1 rstudioapi_0.16.0 knitr_1.47
[49] htmltools_0.5.8.1 rmarkdown_2.27 compiler_4.4.0 getPass_0.2-4