> if (!

requireNamespace("BiocManager", quietly = TRUE))

+
+ install.packages("BiocManager")
>
> BiocManager::install(c("DESeq2", "GEOquery", "ggplot2"))
'getOption("repos")' replaces Bioconductor standard repositories, see
'help("repositories", package = "BiocManager")' for details.
Replacement repositories:
CRAN: https://cran.rstudio.com/
Bioconductor version 3.18 (BiocManager 1.30.22), R 4.3.3 (2024-02-29 ucrt)
Installing package(s) 'DESeq2'
trying URL 'https://bioconductor.org/packages/3.18/bioc/bin/windows/contrib/4.3/
DESeq2_1.42.1.zip'
Content type 'application/zip' length 2396005 bytes (2.3 MB)
downloaded 2.3 MB

package ‘DESeq2’ successfully unpacked and MD5 sums checked

The downloaded binary packages are in

C:\Users\PMYLS\AppData\Local\Temp\RtmpMZmNVd\downloaded_packages
Installation paths not writeable, unable to update packages
path: C:/Program Files/R/R-4.3.3/library
packages:
boot
Old packages: 'bdsmatrix', 'curl', 'data.table', 'digest', 'GenomeInfoDb',
'locfit', 'RcppArmadillo', 'S4Arrays', 'tidyselect'
Update all/some/none? [a/s/n]:
n
Warning message:
package(s) not installed when version(s) same as or greater than current; use
`force = TRUE` to re-install: 'GEOquery' 'ggplot2'
> library(GEOquery)
Loading required package: Biobase
Welcome to Bioconductor

Vignettes contain introductory material; view with

'browseVignettes()'. To cite Bioconductor, see
'citation("Biobase")', and for packages 'citation("pkgname")'.

Setting options('download.file.method.GEOquery'='auto')
Setting options('GEOquery.inmemory.gpl'=FALSE)
> library(DESeq2)
Loading required package: S4Vectors
Loading required package: stats4

Attaching package: ‘S4Vectors’

The following object is masked from ‘package:utils’:

findMatches

The following objects are masked from ‘package:base’:

expand.grid, I, unname
Loading required package: IRanges

Attaching package: ‘IRanges’

The following object is masked from ‘package:grDevices’:

windows

Loading required package: GenomicRanges

Loading required package: GenomeInfoDb
Loading required package: SummarizedExperiment
Loading required package: MatrixGenerics
Loading required package: matrixStats

Attaching package: ‘matrixStats’

The following objects are masked from ‘package:Biobase’:

anyMissing, rowMedians

Attaching package: ‘MatrixGenerics’

The following objects are masked from ‘package:matrixStats’:

colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse,

colCounts, colCummaxs, colCummins, colCumprods, colCumsums,
colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs,
colMads, colMaxs, colMeans2, colMedians, colMins, colOrderStats,
colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds,
colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads,
colWeightedMeans, colWeightedMedians, colWeightedSds,
colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet,
rowCollapse, rowCounts, rowCummaxs, rowCummins, rowCumprods,
rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps,
rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians, rowMins,
rowOrderStats, rowProds, rowQuantiles, rowRanges, rowRanks,
rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs, rowVars,
rowWeightedMads, rowWeightedMeans, rowWeightedMedians,
rowWeightedSds, rowWeightedVars

The following object is masked from ‘package:Biobase’:

rowMedians

> library(ggplot)
Error in library(ggplot) : there is no package called ‘ggplot’
> url="https://www.ncbi.nlm.nih.gov/geo/download/?
acc=GSE152075&format=file&file=GSE152075%5Fraw%5Fcounts%5FGEO%2Etxt%2Egz"
>
> download.file(url, "raw_reads.gz")
trying URL 'https://www.ncbi.nlm.nih.gov/geo/download/?
acc=GSE152075&format=file&file=GSE152075%5Fraw%5Fcounts%5FGEO%2Etxt%2Egz'
Content type 'application/octet-stream' length 7121083 bytes (6.8 MB)
downloaded 6.8 MB
>
> raw_counts <- read.delim("raw_reads.gz", stringsAsFactors=FALSE, sep = " ")
> clindata <- data[["GSE152075_series_matrix.txt.gz"]]@phenoData@data
>
> rownames(clindata) <- clindata$title
>
> head(raw_counts[,c(1:10)])
POS_001 POS_002 POS_003 POS_004 POS_005 POS_006 POS_007 POS_008
A1BG 0 1 0 0 18 8 0 1
A1CF 0 0 2 0 0 0 0 0
A2M 69 36 84 42 83 46 26 0
A2ML1 2 0 0 0 3 30 0 32
A2MP1 0 0 0 0 21 0 0 0
A3GALT2 0 0 0 0 0 0 0 0
POS_009 POS_010
A1BG 0 1
A1CF 0 0
A2M 93 6
A2ML1 6 0
A2MP1 0 0
A3GALT2 0 0
> all(rownames(clindata) %in% colnames(raw_counts))
[1] TRUE
> #the outcome should be TRUE
>
> all(colnames(raw_counts) %in% rownames(clindata))
[1] TRUE
> #the outcome should be TRUE
> colnames(clindata)[colnames(clindata) == "sequencing_batch:ch1"] <- "batch"
> clindata$batch <- as.factor(clindata$batch)
> colnames(clindata)[colnames(clindata) == "n1_ct:ch1"] <- "ct"
> colnames(clindata)[colnames(clindata) == "sars-cov-2 positivity:ch1"] <- "positivity"
> clindata$positivity[clindata$positivity == "pos"] <- "COVID19"
> clindata$positivity[clindata$positivity == "neg"] <- "HEALTHY"
> clindata$positivity <- as.factor(clindata$positivity)
> dds <- DESeqDataSetFromMatrix(countData = raw_counts,
+ colData = clindata,
+ design = formula(~positivity))
>
> dds <- estimateSizeFactors(dds)
>
> sizeFactors(dds)
POS_001 POS_002 POS_003 POS_004 POS_005 POS_006
0.82150765 1.94503946 1.49097573 1.43709833 3.62733595 4.51302810
POS_007 POS_008 POS_009 POS_010 POS_011 POS_012
0.43879900 0.65560763 1.39349809 0.52104453 0.39175741 1.48338323
POS_013 POS_014 POS_015 POS_016 POS_017 POS_018
1.20106632 0.66727757 2.37800300 0.46610178 0.20797773 1.76775711
POS_019 POS_020 POS_021 POS_022 POS_023 POS_024
2.65142274 1.76886420 1.97030866 0.70447111 0.83783570 0.66872019
POS_025 POS_026 POS_027 POS_028 POS_029 POS_030
0.89108814 2.82454001 0.68920670 1.65203141 5.56849704 0.94758147
POS_031 POS_032 POS_033 POS_034 POS_035 POS_036
0.65483358 0.53204324 1.93439648 0.39280125 0.57118414 0.53233011
 POS_037 POS_039 POS_040 POS_041 POS_042 POS_043
0.34756028 0.47397183 0.40659234 0.25446185 0.42860230 0.19437360
POS_044 POS_045 POS_046 POS_047 POS_048 POS_049
0.17275320 1.22474628 0.77871114 0.12095101 0.66709974 0.48064602
POS_050 POS_051 POS_052 POS_053 POS_055 POS_056
1.45778444 2.48142837 0.45258156 0.10172997 0.17053833 1.38160164
POS_057 POS_058 POS_059 POS_060 POS_061 POS_062
1.29123784 0.55072877 0.22723110 0.56090799 0.35834056 0.26984073
POS_063 POS_064 POS_065 POS_066 POS_067 POS_068
0.47723135 0.78893150 2.11919180 0.90699793 0.48525196 1.02668277
POS_069 POS_070 POS_071 POS_072 POS_073 POS_074
1.87880358 2.04558215 2.38657434 1.54185124 2.97612992 0.84452744
POS_075 POS_076 POS_077 POS_078 POS_079 POS_080
7.49339297 1.21680928 1.39623188 1.18920821 1.72370694 3.73009966
POS_081 POS_082 POS_083 POS_084 POS_085 POS_086
0.99115152 0.90122977 0.83929591 3.88800843 0.26679759 1.85159294
POS_087 POS_088 POS_089 POS_090 POS_092 POS_093
2.16993805 6.61679685 0.79220694 0.62224767 1.26100450 0.86008080
POS_094 POS_095 POS_096 POS_097 POS_098 POS_099
0.22813167 0.43758805 0.44854795 1.45678182 0.40660918 1.09052879
POS_100 POS_101 POS_102 POS_103 POS_104 POS_105
1.83335164 3.55862141 0.76396153 0.39301027 1.64352606 1.61382236
POS_106 POS_107 POS_108 POS_109 POS_111 POS_112
0.62527236 1.14875751 0.87637855 5.37132867 1.62077312 0.24354799
POS_113 POS_114 POS_115 POS_116 POS_117 POS_118
3.51304483 2.73965518 1.93933261 2.74366386 0.74948303 0.27978979
POS_119 POS_120 POS_121 POS_122 POS_123 POS_124
0.47153011 0.43800907 0.50394776 2.32665218 0.62008987 2.31793752
POS_125 POS_126 POS_127 POS_128 POS_129 POS_130
0.99220069 2.03392838 0.22334167 0.67704559 2.32969666 0.48883342
POS_131 POS_132 POS_133 POS_134 POS_135 POS_136
0.53514534 0.45261247 0.72774428 0.72577585 0.64276919 3.51469011
POS_137 POS_138 POS_139 POS_141 POS_142 POS_143
0.18067774 1.88115206 1.40814355 0.76515662 1.60223590 0.88027462
POS_144 POS_145 POS_146 POS_147 POS_148 POS_149
1.43963401 0.65839656 0.05540700 0.35236864 0.20813511 1.91410067
POS_150 POS_151 POS_152 POS_153 POS_154 POS_155
0.07650428 0.67487725 0.45105265 0.43339346 1.69155623 0.05019283
POS_156 POS_157 POS_158 POS_159 POS_160 POS_161
0.46078844 0.83930253 0.20510959 1.40537034 1.11178292 7.24019951
POS_162 POS_163 POS_164 POS_165 POS_166 POS_167
0.51851689 1.04121822 0.77355313 1.06525467 0.94935689 2.25489953
POS_168 POS_169 POS_170 POS_171 POS_172 POS_173
0.87628146 0.31268569 0.46983015 1.70957200 0.94406679 1.39192462
POS_174 POS_175 POS_176 POS_177 POS_178 POS_179
3.40383823 1.94774725 0.54670521 0.46047380 0.33685622 0.12765649
POS_180 POS_181 POS_182 POS_183 POS_184 POS_185
0.75606455 3.35223007 0.67548587 2.19360437 0.67183236 2.71454278
POS_186 POS_187 POS_188 POS_189 POS_190 POS_191
0.86494950 1.21993539 0.97910567 2.50798640 0.29825885 0.97967285
POS_192 POS_193 POS_194 POS_195 POS_196 POS_197
0.49169915 0.64665787 1.52663415 0.44409168 0.65953915 1.38778909
POS_198 POS_199 POS_200 POS_201 POS_202 POS_203
1.75246305 0.57304878 0.19125847 1.17950068 0.80035965 1.24036229
POS_204 POS_205 POS_206 POS_207 POS_208 POS_209
0.86344403 1.21692247 0.47680813 2.23865426 0.35572869 2.45584930
POS_210 POS_211 POS_212 POS_213 POS_214 POS_215
1.12638932 0.66480381 0.13646066 1.09477754 1.22109344 1.06264764
POS_216 POS_217 POS_218 POS_219 POS_220 POS_221
0.89569387 4.43385331 1.02481516 1.93410208 0.82905496 0.35154336
POS_222 POS_223 POS_224 POS_225 POS_226 POS_227
0.59248371 1.26566422 1.20728414 0.64244505 1.57791538 0.91876082
POS_228 POS_229 POS_230 POS_231 POS_232 POS_233
5.49855996 3.26113764 2.57206244 0.29095125 4.29115902 0.36134329
POS_234 POS_236 POS_237 POS_238 POS_239 POS_240
0.93756145 0.80413070 4.99509414 0.86380736 3.42879142 0.73288148
POS_241 POS_242 POS_243 POS_244 POS_245 POS_246
1.45869878 1.10855604 0.04013997 0.74576270 1.96759049 1.18448495
POS_247 POS_249 POS_250 POS_251 POS_252 POS_253
3.71692298 11.31606710 2.55147670 1.38604345 3.35309454 0.59140823
POS_254 POS_255 POS_256 POS_257 POS_258 POS_259
1.26495581 0.46507632 1.56513781 1.12561370 1.88594837 0.29517603
POS_260 POS_261 POS_262 POS_263 POS_264 POS_265
2.25030827 1.87961440 1.22437654 0.12453718 1.23359623 0.33468218
POS_266 POS_267 POS_268 POS_269 POS_270 POS_271
0.34376771 1.71780175 0.17010155 0.79511621 0.35210654 0.96247569
POS_272 POS_273 POS_274 POS_275 POS_276 POS_277
0.85895689 0.25033812 0.71824493 1.04186945 0.73034481 0.27359535
POS_278 POS_279 POS_280 POS_281 POS_282 POS_283
0.85724564 1.33676511 1.20124727 0.35374233 0.70733949 0.38453206
POS_284 POS_285 POS_286 POS_287 POS_288 POS_289
2.20831594 0.57290557 0.66315359 0.58636365 1.32666929 0.88244054
POS_290 POS_291 POS_292 POS_293 POS_295 POS_296
1.68949980 0.97307998 0.18268736 1.92722988 1.21571762 0.52147981
POS_297 POS_298 POS_299 POS_300 POS_301 POS_302
0.45089568 0.76762646 0.53159548 2.48967051 1.01910535 1.90993648
POS_303 POS_304 POS_305 POS_306 POS_307 POS_308
0.90335967 9.48393988 0.86630680 25.10848779 6.55766749 1.08771222
POS_309 POS_310 POS_311 POS_312 POS_313 POS_314
0.52786853 1.26456746 4.13002700 2.21255423 2.32132398 0.29894207
POS_315 POS_316 POS_317 POS_318 POS_319 POS_320
0.73862324 0.88613108 4.55930033 1.11748432 3.22116909 0.39079219
POS_321 POS_322 POS_323 POS_324 POS_325 POS_326
1.09513668 1.16927703 1.85012412 0.18985578 0.66196868 0.81364638
POS_327 POS_328 POS_329 POS_330 POS_331 POS_332
0.54969286 0.37761658 0.20494907 0.27667501 0.35714040 0.75757113
POS_333 POS_334 POS_335 POS_336 POS_337 POS_338
2.01587102 1.40169759 0.26393964 0.09279402 0.97126945 0.19179572
POS_339 POS_340 POS_341 POS_342 POS_343 POS_344
1.95474753 1.25568854 0.93456175 0.77785778 2.56274842 0.68100943
POS_345 POS_346 POS_347 POS_348 POS_349 POS_350
0.76437481 1.21416099 0.63417381 0.77094834 0.52882066 1.73103239
POS_351 POS_352 POS_353 POS_354 POS_355 POS_356
0.49109973 0.76349447 1.46427434 5.94220105 3.72030765 2.99336232
POS_357 POS_358 POS_359 POS_360 POS_361 POS_362
1.95199067 4.24926149 2.11426671 2.78373530 2.66847148 2.15866379
POS_363 POS_364 POS_365 POS_366 POS_367 POS_368
0.99813337 0.58707444 3.38615020 9.02672812 1.51376821 1.76615253
POS_369 POS_370 POS_371 POS_372 POS_373 POS_374
1.78379467 4.12942189 4.40204804 0.31802532 4.85824628 1.04779954
 POS_375 POS_376 POS_377 POS_378 POS_379 POS_380
0.91546325 1.48488204 0.79519625 2.16997799 0.49333296 1.46388772
POS_381 POS_382 POS_383 POS_384 POS_385 POS_386
1.40262413 0.45137633 2.65718673 0.66674382 0.61703304 3.41946321
POS_387 POS_388 POS_389 POS_390 POS_391 POS_392
3.73637422 1.89777620 4.19211188 1.62392700 0.58213783 1.92832592
POS_393 POS_394 POS_395 POS_396 POS_397 POS_398
2.22045302 1.30687631 1.27050728 1.23931685 3.63717596 1.92744878
POS_399 POS_400 POS_401 POS_402 POS_403 POS_404
1.84911278 1.30140569 1.93450167 0.50514736 1.29997647 0.59868617
POS_405 POS_406 POS_407 POS_408 POS_409 POS_410
1.67579408 1.86980149 1.69006956 5.61899295 2.10884621 0.86825111
POS_411 POS_412 POS_413 POS_414 POS_415 POS_416
9.62551434 1.56271146 2.75351885 0.96724788 1.64361125 0.25153628
POS_417 POS_418 POS_419 POS_420 POS_421 POS_422
1.57934432 0.54330843 0.44028926 0.62898260 0.83088379 0.33791812
POS_423 POS_424 POS_425 POS_426 POS_427 POS_428
1.20302976 0.22366177 0.40970666 0.40435984 0.45548472 0.61097903
POS_429 POS_430 POS_431 POS_432 POS_433 POS_434
1.73742817 3.57972740 2.61260389 3.66782342 1.14744774 0.59514112
POS_435 POS_436 POS_437 POS_438 NEG_001 NEG_002
0.89266608 1.12552906 2.34820749 1.77915572 1.37361248 0.99191586
NEG_003 NEG_004 NEG_005 NEG_006 NEG_007 NEG_008
2.50944161 1.30918020 0.71934137 1.03537857 1.66126743 0.44623436
NEG_009 NEG_010 NEG_011 NEG_012 NEG_013 NEG_014
0.95245375 2.53031524 1.50412382 2.25674763 1.06646617 6.31751393
NEG_016 NEG_018 NEG_019 NEG_020 NEG_021 NEG_022
2.41952118 0.17048801 0.16396356 0.63613827 0.19168077 1.60343400
NEG_023 NEG_024 NEG_025 NEG_026 NEG_027 NEG_028
0.44193872 12.66897032 0.93038627 0.68512621 0.55755191 0.08636394
NEG_030 NEG_031 NEG_032 NEG_035 NEG_036 NEG_037
0.43001183 0.25335642 0.38799176 0.89077939 0.28143092 1.96180106
NEG_038 NEG_039 NEG_040 NEG_042 NEG_043 NEG_046
2.02589805 33.84796729 4.22997327 7.59535344 4.35877734 0.18902771
NEG_047 NEG_048 NEG_049 NEG_050 NEG_051 NEG_052
0.42208111 1.51360842 0.83139662 1.37211524 2.61779054 0.93404869
NEG_054 NEG_055 NEG_056 NEG_057 NEG_058 NEG_059
1.14946016 0.95652615 0.87371970 3.62714412 0.78428673 1.56996972
NEG_060 NEG_062 NEG_063 NEG_065
0.24250539 1.00778315 1.26921554 1.33090346
> dds <- DESeq(dds)
using pre-existing size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
-- replacing outliers and refitting for 7069 genes
-- DESeq argument 'minReplicatesForReplace' = 7
-- original counts are preserved in counts(dds)
estimating dispersions
fitting model and testing
>
> res <- results(dds)
>
> summary(res)

out of 31474 with nonzero total read count

adjusted p-value < 0.1
LFC > 0 (up) : 4923, 16%
LFC < 0 (down) : 1582, 5%
outliers [1] : 0, 0%
low counts [2] : 7308, 23%
(mean count < 0)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

>
> plotMA(res)