Normalization performance report
- study: Illumina methylation data
- author: Analyst
- date: 28 agosto, 2023
Parameters used to test normalization
## $variables
## [1] "Slide" "Sex" "Age" "Smoking"
##
## $control.pcs
## [1] 1 2 3 4 5
##
## $batch.pcs
## [1] 1 2 3 4 5
##
## $batch.threshold
## [1] 0.01
##
## $colours
## NULL
Control probe scree plots
The variance captured by each principal component.
Principal components of the control probes
The following plots show the first 3 principal components of the control matrix colored by batch variables. Batch variables with more than 10 levels are omitted.
Control probe associations with measured batch variables
Principal components of the control probes were regressed against batch variables. Shown are the \(-log_{10}\) p-values for these regressions. The horizontal dotted line denotes \(p = 0.05\) in log-scale.
The following plots show regression coefficients when each principal component is regressed against each batch variable level along with 95% confidence intervals. Cases significantly different from zero are coloured red (p < 0.01, t-test).
| batch.variable | batch.value | principal.component | test | p.value | estimate | lower | upper | r2 |
|---|---|---|---|---|---|---|---|---|
| Slide | PC1 | F-test | 3.02e-13 | 4.137 | 0.4288 | |||
| Slide | 7800246140 | PC1 | t-test | 1.09e-03 | -5.375 | -9.033 | -1.717 | 0.0359 |
| Slide | 7796814016 | PC1 | t-test | 9.08e-05 | 6.098 | 2.650 | 9.546 | 0.0512 |
| Slide | 5765205058 | PC1 | t-test | 7.62e-03 | 4.978 | 0.818 | 9.137 | 0.0241 |
| Slide | 5765205059 | PC1 | t-test | 9.73e-06 | 8.164 | 4.092 | 12.236 | 0.0649 |
| Slide | 5730192048 | PC1 | t-test | 6.36e-05 | 5.948 | 2.658 | 9.237 | 0.0534 |
| Slide | 5730053041 | PC1 | t-test | 9.29e-04 | 6.157 | 2.024 | 10.289 | 0.0369 |
| Age | 57 | PC1 | t-test | 3.35e-03 | 5.465 | 1.315 | 9.614 | 0.0291 |
| Slide | PC2 | F-test | 6.52e-72 | 26.578 | 0.8283 | |||
| Slide | 7800246087 | PC2 | t-test | 6.01e-04 | -2.069 | -3.407 | -0.731 | 0.0427 |
| Slide | 7800246140 | PC2 | t-test | 5.42e-05 | -2.537 | -3.926 | -1.148 | 0.0583 |
| Slide | 7800246046 | PC2 | t-test | 1.75e-03 | 1.797 | 0.520 | 3.073 | 0.0357 |
| Slide | 7512560115 | PC2 | t-test | 8.36e-03 | 1.518 | 0.235 | 2.802 | 0.0255 |
| Slide | 5730192053 | PC2 | t-test | 3.73e-03 | 2.137 | 0.496 | 3.777 | 0.0307 |
| Slide | 5765205058 | PC2 | t-test | 1.49e-03 | -2.168 | -3.685 | -0.651 | 0.0368 |
| Slide | 5730053006 | PC2 | t-test | 2.85e-14 | -7.313 | -9.358 | -5.267 | 0.1912 |
| Slide | 5730053010 | PC2 | t-test | 3.12e-19 | -8.698 | -10.719 | -6.678 | 0.2546 |
| Slide | 5730053011 | PC2 | t-test | 2.02e-08 | -7.348 | -10.205 | -4.491 | 0.1094 |
| Slide | 5730053027 | PC2 | t-test | 9.87e-20 | -8.839 | -10.860 | -6.819 | 0.2608 |
| Slide | 5730053037 | PC2 | t-test | 1.11e-12 | -6.721 | -8.744 | -4.698 | 0.1690 |
| Slide | 5730192036 | PC2 | t-test | 3.39e-03 | -2.360 | -4.153 | -0.566 | 0.0314 |
| Slide | 5730192048 | PC2 | t-test | 1.44e-16 | -4.511 | -5.660 | -3.362 | 0.2215 |
| Slide | 5730192057 | PC2 | t-test | 7.68e-05 | -3.531 | -5.506 | -1.555 | 0.0564 |
| Slide | 5730053048 | PC2 | t-test | 1.18e-04 | -2.615 | -4.119 | -1.112 | 0.0535 |
| Sex | M | PC2 | t-test | 9.09e-04 | -1.074 | -1.785 | -0.364 | 0.0382 |
| Age | 55 | PC2 | t-test | 1.48e-03 | -1.755 | -2.982 | -0.529 | 0.0364 |
| Age | 69 | PC2 | t-test | 7.19e-04 | -1.902 | -3.150 | -0.654 | 0.0411 |
| Age | 62 | PC2 | t-test | 5.05e-03 | -1.473 | -2.642 | -0.304 | 0.0286 |
| Age | 48 | PC2 | t-test | 8.38e-04 | -2.578 | -4.292 | -0.863 | 0.0403 |
| Age | 24 | PC2 | t-test | 6.53e-04 | -6.196 | -10.236 | -2.157 | 0.0420 |
| Smoking | never | PC2 | t-test | 7.38e-04 | -0.947 | -1.560 | -0.333 | 0.0398 |
| Slide | PC3 | F-test | 1.19e-24 | 6.959 | 0.5581 | |||
| Slide | 5730192053 | PC3 | t-test | 6.01e-04 | -2.381 | -3.923 | -0.840 | 0.0401 |
| Slide | 5765205058 | PC3 | t-test | 2.96e-10 | -4.166 | -5.599 | -2.733 | 0.1277 |
| Slide | 5765205059 | PC3 | t-test | 4.30e-11 | -4.182 | -5.552 | -2.812 | 0.1399 |
| Slide | 5730053006 | PC3 | t-test | 2.94e-03 | 2.526 | 0.634 | 4.417 | 0.0303 |
| Slide | 5730192048 | PC3 | t-test | 3.12e-07 | -2.613 | -3.733 | -1.494 | 0.0870 |
| Slide | 5730053048 | PC3 | t-test | 1.78e-03 | -2.014 | -3.449 | -0.580 | 0.0334 |
| Age | 65 | PC3 | t-test | 7.50e-03 | -2.037 | -3.737 | -0.338 | 0.0246 |
| Slide | PC4 | F-test | 1.90e-65 | 22.811 | 0.8054 | |||
| Slide | 7800246087 | PC4 | t-test | 2.77e-03 | -1.628 | -2.840 | -0.417 | 0.0303 |
| Slide | 7800246140 | PC4 | t-test | 2.38e-05 | -2.282 | -3.475 | -1.089 | 0.0596 |
| Slide | 7800246006 | PC4 | t-test | 5.98e-03 | 1.692 | 0.320 | 3.065 | 0.0257 |
| Slide | 7800246054 | PC4 | t-test | 6.68e-03 | 1.478 | 0.264 | 2.693 | 0.0250 |
| Slide | 7800246034 | PC4 | t-test | 4.43e-03 | 1.888 | 0.410 | 3.367 | 0.0275 |
| Slide | 7800246046 | PC4 | t-test | 2.73e-04 | 1.875 | 0.733 | 3.018 | 0.0446 |
| Slide | 7800246057 | PC4 | t-test | 2.57e-03 | 1.853 | 0.485 | 3.222 | 0.0308 |
| Slide | 7512560103 | PC4 | t-test | 1.92e-03 | -1.604 | -2.753 | -0.454 | 0.0326 |
| Slide | 7512560104 | PC4 | t-test | 7.67e-03 | -1.539 | -2.825 | -0.252 | 0.0242 |
| Slide | 7512560115 | PC4 | t-test | 9.09e-03 | -1.352 | -2.507 | -0.196 | 0.0232 |
| Slide | 7766130166 | PC4 | t-test | 1.07e-03 | -2.683 | -4.507 | -0.860 | 0.0361 |
| Slide | 5730053006 | PC4 | t-test | 3.46e-03 | 2.367 | 0.563 | 4.170 | 0.0290 |
| Slide | 5730053010 | PC4 | t-test | 3.90e-04 | 2.861 | 1.070 | 4.652 | 0.0424 |
| Slide | 5730053027 | PC4 | t-test | 5.08e-03 | 2.270 | 0.464 | 4.075 | 0.0267 |
| Slide | 5730053037 | PC4 | t-test | 8.70e-06 | 3.566 | 1.797 | 5.335 | 0.0658 |
| Slide | 5730192057 | PC4 | t-test | 6.26e-04 | -2.761 | -4.555 | -0.967 | 0.0395 |
| Slide | 5730053048 | PC4 | t-test | 3.19e-05 | -2.540 | -3.889 | -1.190 | 0.0578 |
| Slide | PC5 | F-test | 3.17e-57 | 18.624 | 0.7717 | |||
| Slide | 7800246085 | PC5 | t-test | 4.02e-03 | -1.103 | -1.958 | -0.249 | 0.0281 |
| Slide | 7800246087 | PC5 | t-test | 1.62e-04 | -1.359 | -2.158 | -0.561 | 0.0478 |
| Slide | 7800246123 | PC5 | t-test | 1.08e-06 | -1.657 | -2.403 | -0.910 | 0.0786 |
| Slide | 7800246018 | PC5 | t-test | 5.00e-03 | -0.923 | -1.656 | -0.191 | 0.0268 |
| Slide | 7800246054 | PC5 | t-test | 9.30e-04 | -1.196 | -1.999 | -0.394 | 0.0370 |
| Slide | 7512560104 | PC5 | t-test | 2.49e-04 | 1.399 | 0.552 | 2.245 | 0.0451 |
| Slide | 7512560115 | PC5 | t-test | 5.48e-05 | 1.379 | 0.623 | 2.135 | 0.0545 |
| Slide | 7512560124 | PC5 | t-test | 2.15e-03 | 1.055 | 0.290 | 1.820 | 0.0319 |
| Slide | 5765205058 | PC5 | t-test | 2.81e-09 | 2.414 | 1.530 | 3.299 | 0.1141 |
| Slide | 5765205059 | PC5 | t-test | 9.81e-05 | 1.585 | 0.684 | 2.486 | 0.0509 |
| Slide | 5730192036 | PC5 | t-test | 1.82e-05 | 2.051 | 0.994 | 3.107 | 0.0613 |
| Slide | 5730192048 | PC5 | t-test | 1.28e-04 | -1.252 | -1.976 | -0.528 | 0.0492 |
| Sex | PC5 | F-test | 6.68e-04 | 11.829 | 0.0389 | |||
| Sex | M | PC5 | t-test | 3.41e-04 | 0.493 | 0.191 | 0.795 | 0.0432 |
| Sex | F | PC5 | t-test | 6.68e-04 | -0.476 | -0.773 | -0.179 | 0.0389 |
Principal components of the normalized betas
The following plots show the first 3 principal components of the most variable probes colored by batch variables. Batch variables with more than 10 levels are omitted.
Normalized probe associations with measured batch variables
The most variable normalized probes were extracted, decomposed into principal components and each component regressed against each batch variable. If the normalization has performed well then there will be no associations between normalized probe PCs and batch variables. Horizontal dotted line denotes \(p = 0.05\) in log-scale.
The following plots show regression coefficients when each principal component is regressed against each batch variable level along with 95% confidence intervals. Cases significantly different from zero are coloured red (p < 0.01, t-test).
| batch.variable | batch.value | principal.component | test | p.value | estimate | lower | upper | r2 |
|---|---|---|---|---|---|---|---|---|
| Slide | PC2 | F-test | 2.90e-36 | 10.38 | 0.6532 | |||
| Slide | 7512560104 | PC2 | t-test | 2.42e-03 | -3.28 | -5.687 | -0.875 | 0.0331 |
| Slide | 5765205058 | PC2 | t-test | 2.80e-04 | 4.18 | 1.628 | 6.727 | 0.0471 |
| Slide | 5730053006 | PC2 | t-test | 2.78e-11 | 10.55 | 7.133 | 13.964 | 0.1476 |
| Slide | 5730053010 | PC2 | t-test | 2.35e-12 | 11.03 | 7.654 | 14.406 | 0.1629 |
| Slide | 5730053011 | PC2 | t-test | 4.90e-06 | 9.92 | 5.134 | 14.696 | 0.0732 |
| Slide | 5730053027 | PC2 | t-test | 3.29e-11 | 10.37 | 6.999 | 13.742 | 0.1471 |
| Slide | 5730053037 | PC2 | t-test | 5.31e-11 | 10.38 | 6.963 | 13.790 | 0.1437 |
| Slide | 5730192048 | PC2 | t-test | 1.18e-10 | 5.91 | 3.929 | 7.889 | 0.1403 |
| Sex | F | PC2 | t-test | 2.48e-03 | 1.37 | 0.412 | 2.333 | 0.0322 |
| Age | 48 | PC2 | t-test | 2.09e-03 | 4.00 | 1.108 | 6.896 | 0.0338 |
| Age | 28 | PC2 | t-test | 7.95e-03 | 5.69 | 0.907 | 10.471 | 0.0254 |
| Smoking | PC2 | F-test | 2.87e-03 | 5.97 | 0.0394 | |||
| Smoking | never | PC2 | t-test | 8.43e-03 | 1.17 | 0.194 | 2.137 | 0.0243 |
| Smoking | former | PC2 | t-test | 2.83e-03 | 1.26 | 0.336 | 2.180 | 0.0312 |
| Slide | PC3 | F-test | 6.47e-04 | 1.96 | 0.2626 | |||
| Slide | 7512560115 | PC3 | t-test | 2.05e-04 | 3.48 | 1.401 | 5.551 | 0.0465 |
| Slide | 7512560124 | PC3 | t-test | 2.04e-04 | 3.52 | 1.420 | 5.622 | 0.0464 |
| Slide | 7512560128 | PC3 | t-test | 1.34e-03 | 3.36 | 1.030 | 5.683 | 0.0349 |
| Slide | 5730053010 | PC3 | t-test | 1.30e-03 | -4.73 | -7.994 | -1.457 | 0.0351 |
| Slide | 5730192048 | PC3 | t-test | 3.52e-03 | -2.62 | -4.621 | -0.622 | 0.0290 |
| Age | PC3 | F-test | 3.01e-05 | 2.28 | 0.2981 | |||
| Age | 69 | PC3 | t-test | 5.40e-04 | -2.97 | -4.879 | -1.066 | 0.0405 |
| Age | 36 | PC3 | t-test | 1.37e-03 | 4.22 | 1.287 | 7.144 | 0.0348 |
| Age | 70 | PC3 | t-test | 6.41e-03 | -4.01 | -7.300 | -0.730 | 0.0253 |
| Age | 46 | PC3 | t-test | 3.69e-03 | 4.93 | 1.145 | 8.707 | 0.0287 |
| Age | 64 | PC3 | t-test | 7.86e-03 | -3.05 | -5.604 | -0.491 | 0.0240 |
| Age | 29 | PC3 | t-test | 4.23e-03 | 8.48 | 1.870 | 15.093 | 0.0278 |
| Slide | PC4 | F-test | 2.31e-03 | 1.82 | 0.2479 | |||
| Slide | 7512560103 | PC4 | t-test | 4.50e-03 | -2.25 | -4.019 | -0.487 | 0.0275 |
| Slide | 5730053041 | PC4 | t-test | 7.74e-05 | -3.70 | -5.767 | -1.626 | 0.0525 |
| Slide | PC5 | F-test | 5.75e-68 | 24.23 | 0.8147 | |||
| Slide | 7800246087 | PC5 | t-test | 2.94e-06 | 3.58 | 1.896 | 5.270 | 0.0727 |
| Slide | 7800246123 | PC5 | t-test | 1.87e-07 | 3.78 | 2.191 | 5.367 | 0.0896 |
| Slide | 7800246137 | PC5 | t-test | 2.47e-03 | 2.13 | 0.564 | 3.694 | 0.0311 |
| Slide | 7800246140 | PC5 | t-test | 1.74e-03 | 2.42 | 0.703 | 4.147 | 0.0333 |
| Slide | 7512560104 | PC5 | t-test | 1.67e-03 | -2.58 | -4.401 | -0.753 | 0.0335 |
| Slide | 7512560124 | PC5 | t-test | 5.23e-04 | 2.55 | 0.918 | 4.178 | 0.0407 |
| Slide | 5730192053 | PC5 | t-test | 3.29e-07 | -4.75 | -6.792 | -2.709 | 0.0861 |
| Slide | 5730504015 | PC5 | t-test | 8.89e-03 | -3.48 | -6.451 | -0.512 | 0.0234 |
| Slide | 5765205058 | PC5 | t-test | 5.99e-06 | -3.97 | -5.910 | -2.039 | 0.0681 |
| Slide | 5765205059 | PC5 | t-test | 3.52e-09 | -5.13 | -7.019 | -3.238 | 0.1131 |
| Slide | 5730192036 | PC5 | t-test | 6.25e-05 | -4.11 | -6.380 | -1.837 | 0.0539 |
| Slide | 5730053041 | PC5 | t-test | 1.74e-03 | -2.74 | -4.687 | -0.793 | 0.0333 |
| Slide | 5730053048 | PC5 | t-test | 3.95e-04 | -3.09 | -5.031 | -1.156 | 0.0424 |
| Sex | PC5 | F-test | 4.66e-14 | 62.90 | 0.1772 | |||
| Sex | M | PC5 | t-test | 7.71e-15 | -2.23 | -2.835 | -1.628 | 0.1877 |
| Sex | F | PC5 | t-test | 1.70e-13 | 2.13 | 1.541 | 2.723 | 0.1706 |
| Age | 33 | PC5 | t-test | 1.82e-03 | 2.94 | 0.842 | 5.041 | 0.0330 |
| Age | 65 | PC5 | t-test | 7.56e-03 | -2.76 | -5.069 | -0.456 | 0.0243 |
| Age | 68 | PC5 | t-test | 3.86e-04 | -2.81 | -4.573 | -1.055 | 0.0423 |
R session information
## R version 4.2.1 (2022-06-23 ucrt)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## Running under: Windows 10 x64 (build 22621)
##
## Matrix products: default
##
## locale:
## [1] LC_COLLATE=Spanish_Spain.utf8 LC_CTYPE=Spanish_Spain.utf8 LC_MONETARY=Spanish_Spain.utf8 LC_NUMERIC=C
## [5] LC_TIME=Spanish_Spain.utf8
##
## attached base packages:
## [1] parallel stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] meffil_1.3.6 preprocessCore_1.58.0 SmartSVA_0.1.3 RSpectra_0.16-1 isva_1.9 JADE_2.0-3
## [7] qvalue_2.28.0 gdsfmt_1.32.0 statmod_1.5.0 quadprog_1.5-8 DNAcopy_1.70.0 fastICA_1.2-3
## [13] lme4_1.1-30 Matrix_1.5-1 multcomp_1.4-20 TH.data_1.1-1 survival_3.3-1 mvtnorm_1.1-3
## [19] matrixStats_0.62.0 markdown_1.7 gridExtra_2.3 Cairo_1.6-0 knitr_1.43 reshape2_1.4.4
## [25] plyr_1.8.7 ggplot2_3.4.2 sva_3.44.0 BiocParallel_1.30.3 genefilter_1.78.0 mgcv_1.8-40
## [31] nlme_3.1-157 limma_3.52.4 sandwich_3.0-2 lmtest_0.9-40 zoo_1.8-11 MASS_7.3-57
## [37] illuminaio_0.38.0 bookdown_0.34
##
## loaded via a namespace (and not attached):
## [1] minqa_1.2.4 colorspace_2.0-3 XVector_0.36.0 fs_1.5.2 base64_2.0.1
## [6] clue_0.3-64 rstudioapi_0.14 farver_2.1.1 bit64_4.0.5 AnnotationDbi_1.58.0
## [11] fansi_1.0.3 xml2_1.3.3 codetools_0.2-19 splines_4.2.1 downlit_0.4.3
## [16] cachem_1.0.6 jsonlite_1.8.3 nloptr_2.0.3 annotate_1.74.0 cluster_2.1.3
## [21] png_0.1-7 compiler_4.2.1 httr_1.4.6 fastmap_1.1.0 cli_3.6.1
## [26] htmltools_0.5.5 tools_4.2.1 gtable_0.3.3 glue_1.6.2 GenomeInfoDbData_1.2.8
## [31] dplyr_1.1.2 Rcpp_1.0.9 Biobase_2.56.0 jquerylib_0.1.4 vctrs_0.6.3
## [36] Biostrings_2.64.1 xfun_0.39 stringr_1.5.0 mime_0.12 lifecycle_1.0.3
## [41] XML_3.99-0.10 edgeR_3.38.4 zlibbioc_1.42.0 scales_1.2.1 yaml_2.3.5
## [46] memoise_2.0.1 sass_0.4.6 stringi_1.7.6 RSQLite_2.2.17 highr_0.10
## [51] S4Vectors_0.34.0 BiocGenerics_0.42.0 boot_1.3-28.1 GenomeInfoDb_1.32.4 commonmark_1.9.0
## [56] rlang_1.1.1 pkgconfig_2.0.3 bitops_1.0-7 evaluate_0.21 lattice_0.20-45
## [61] labeling_0.4.2 bit_4.0.4 tidyselect_1.2.0 magrittr_2.0.3 R6_2.5.1
## [66] IRanges_2.30.1 generics_0.1.3 DBI_1.1.3 pillar_1.9.0 withr_2.5.0
## [71] KEGGREST_1.36.3 RCurl_1.98-1.7 tibble_3.2.1 crayon_1.5.2 utf8_1.2.2
## [76] rmarkdown_2.23 locfit_1.5-9.6 grid_4.2.1 blob_1.2.4 digest_0.6.29
## [81] xtable_1.8-4 openssl_2.0.3 stats4_4.2.1 munsell_0.5.0 bslib_0.5.0
## [86] askpass_1.1