Here we will show a very basic example how to use grandR to perform differential gene expression analysis. For much more vignettes, see the grandR website.
We will use data from [1]. These are SLAM-seq data from multiple time points (1h,2h,4h,6h and 20h) after acute depletion of BANP. BANP is a transcriptional activator that binds to unmethylated CGCG motifs in CpG island promoters.
We first load the grandR package and the read the GRAND-SLAM output table directly from zenodo:
suppressPackageStartupMessages({
library(grandR)
library(ggplot2)
library(patchwork)
})
data <- ReadGRAND("https://zenodo.org/record/6976391/files/BANP.tsv.gz",
design=c("Cell","Experimental.time","Genotype",Design$dur.4sU,Design$has.4sU,Design$Replicate))The GRAND-SLAM output contains any gene with at least 1 read, so let’s filter this (by default: at least 100 reads in half of the samples)
data <- FilterGenes(data)Let’s do a quick quality control and check the principal component
analysis. We have to setup the Condition first to get nice
colors:
Condition(data) <- c("Genotype","Experimental.time.original","has.4sU")
PlotPCA(data)Now we perform differential gene expression analysis:
# differential expression, see differential expression vignette
contrasts <- GetContrasts(data,contrast=c("Experimental.time.original","0h"),columns = Genotype=='dTag')
data <- LFC(data,name.prefix = "total",contrasts = contrasts)
data <- PairwiseDESeq2(data,name.prefix = "total",contrasts = contrasts)
data <- LFC(data,name.prefix = "new",contrasts = contrasts,mode="new", normalization = "total")
data <- PairwiseDESeq2(data,name.prefix = "new",contrasts = contrasts,mode="new", normalization = "total")The MA plots indicate that changes in new RNA are much more pronounced than changes in total RNA, even after 4h:
MAPlot(data,analysis = "total.4h vs 0h")|
MAPlot(data,analysis = "new.4h vs 0h")We can easily obtain significantly differentially regulated genes (by default: Q<0.05 & abs(LFC)>=1):
genes <- GetSignificantGenes(data,analysis = "new.4h vs 0h", criteria = Q<0.05 & LFC< -1)
GetAnalysisTable(data,analyses = "4h vs 0h",genes = genes, columns = "LFC|Q", gene.info = FALSE)| total.4h vs 0h.LFC | total.4h vs 0h.Q | new.4h vs 0h.LFC | new.4h vs 0h.Q | |
|---|---|---|---|---|
| Bzw1 | -0.4049167 | 0.0000000 | -1.591874 | 0.0000000 |
| Cep350 | -0.9428386 | 0.0000000 | -1.310057 | 0.0000000 |
| Ptpn4 | -0.8717413 | 0.0000000 | -1.290797 | 0.0000000 |
| Pign | -0.7468109 | 0.0000000 | -1.740792 | 0.0000000 |
| Pfkfb2 | -1.2109515 | 0.0000000 | -2.034935 | 0.0197814 |
| Rpl7a | -0.2087491 | 0.0213640 | -1.433379 | 0.0000000 |
| 2310035C23Rik | -0.7708118 | 0.0000000 | -1.127441 | 0.0000000 |
| Dars | -0.1614027 | 0.0205584 | -1.712862 | 0.0000000 |
| Lrsam1 | -1.1848762 | 0.0000000 | -1.532849 | 0.0007304 |
| Casc4 | -0.6368342 | 0.0000000 | -1.684313 | 0.0000001 |
| Rc3h2 | -1.1937913 | 0.0000000 | -1.371584 | 0.0000000 |
| March7 | -1.1202654 | 0.0000000 | -1.366951 | 0.0000000 |
| Gm9833 | -0.8112235 | 0.0000000 | -2.778287 | 0.0000000 |
| Zfp512b | -1.2073193 | 0.0000000 | -1.747552 | 0.0000000 |
| Myef2 | -0.9035457 | 0.0000000 | -3.590065 | 0.0000000 |
| Lcmt2 | -1.4894524 | 0.0000000 | -1.684084 | 0.0000006 |
| Cenpb | -1.2772139 | 0.0000000 | -2.531545 | 0.0000000 |
| Wdr77 | -1.3261003 | 0.0000000 | -3.020690 | 0.0000002 |
| Atp5f1 | -0.1750994 | 0.0682100 | -1.588790 | 0.0000033 |
| Nmd3 | -0.6177977 | 0.0000000 | -1.046226 | 0.0000000 |
| Rbm39 | -0.9881069 | 0.0000000 | -1.628205 | 0.0000000 |
| Snx5 | -0.2761889 | 0.0000283 | -1.045626 | 0.0000000 |
| Smg5 | -1.4243152 | 0.0000000 | -2.140475 | 0.0000000 |
| Ttc14 | -0.8866021 | 0.0000000 | -1.279095 | 0.0000000 |
| Strip1 | -1.1181880 | 0.0000000 | -3.584319 | 0.0000000 |
| Rnpc3 | -1.0144526 | 0.0000000 | -1.906450 | 0.0000000 |
| Mms22l | -0.6410693 | 0.0000000 | -1.204826 | 0.0000000 |
| Agl | -0.6282185 | 0.0000000 | -1.724997 | 0.0020372 |
| Srsf10 | -0.6756414 | 0.0000000 | -1.041562 | 0.0000000 |
| Rraga | -1.1960975 | 0.0000000 | -2.626356 | 0.0000043 |
| Prdx1 | -0.1498166 | 0.0546104 | -1.248716 | 0.0000000 |
| Minos1 | -0.2891798 | 0.0682515 | -1.398761 | 0.0015429 |
| Asph | -0.3565418 | 0.0000000 | -1.681709 | 0.0000000 |
| Pole | -0.5164748 | 0.0000000 | -1.583460 | 0.0000000 |
| Atraid | -1.1965615 | 0.0000000 | -2.183169 | 0.0021098 |
| Mob1b | -1.9374683 | 0.0000000 | -2.557860 | 0.0000000 |
| Ncapg | -0.4715520 | 0.0000000 | -1.524758 | 0.0000000 |
| Nop14 | -0.7061881 | 0.0000000 | -1.231487 | 0.0000000 |
| Denr | -0.7719097 | 0.0000000 | -3.558633 | 0.0046419 |
| Mdh2 | -0.4494444 | 0.0000019 | -2.842117 | 0.0000165 |
| Ap4m1 | -0.6352704 | 0.0000000 | -1.005392 | 0.0000081 |
| Chst12 | -1.7593868 | 0.0000000 | -1.787141 | 0.0029205 |
| Rfc5 | -0.4190605 | 0.0000000 | -1.263439 | 0.0000000 |
| Slc5a6 | -1.0656930 | 0.0000000 | -1.460581 | 0.0000029 |
| Mob1a | -1.7499198 | 0.0000000 | -2.223078 | 0.0000000 |
| Mad1l1 | -0.7541517 | 0.0000000 | -3.045967 | 0.0000000 |
| Fgfr1op2 | -0.8360517 | 0.0000000 | -1.123028 | 0.0003901 |
| Ergic2 | -0.7849733 | 0.0000000 | -1.593984 | 0.0000000 |
| Srrt | -0.5493759 | 0.0000000 | -1.134517 | 0.0000000 |
| Tmem209 | -0.9769144 | 0.0000000 | -1.131132 | 0.0000000 |
| Tubgcp5 | -1.7178942 | 0.0000000 | -3.865639 | 0.0008877 |
| Crebzf | -1.0726153 | 0.0000000 | -1.093507 | 0.0000000 |
| Supt5 | -0.5697177 | 0.0000000 | -1.342800 | 0.0000000 |
| Arnt2 | -0.3207844 | 0.0395396 | -1.426045 | 0.0090770 |
| Edrf1 | -1.5350037 | 0.0000000 | -2.081083 | 0.0000000 |
| Btbd10 | -1.6118774 | 0.0000000 | -2.086394 | 0.0027987 |
| Zfp941 | -0.9880679 | 0.0000000 | -1.145216 | 0.0000000 |
| Ikzf5 | -0.9380561 | 0.0000000 | -1.023575 | 0.0000132 |
| Upf3a | -0.7774655 | 0.0000000 | -2.507884 | 0.0000000 |
| Micu3 | -2.0381825 | 0.0000000 | -3.451641 | 0.0000000 |
| Arl2bp | -0.5145906 | 0.0000000 | -1.308256 | 0.0374124 |
| Tcf25 | -0.3281309 | 0.0000000 | -1.107098 | 0.0000747 |
| Slc25a4 | -0.2832186 | 0.0004546 | -1.364629 | 0.0000083 |
| Zfp317 | -1.8563560 | 0.0000000 | -2.046163 | 0.0000000 |
| Ddhd2 | -1.4304835 | 0.0000000 | -3.523651 | 0.0216512 |
| Ankrd49 | -1.0487246 | 0.0000000 | -1.201137 | 0.0000000 |
| Ciapin1 | -0.5332762 | 0.0000000 | -1.169674 | 0.0000000 |
| Ift46 | -0.5200090 | 0.0000000 | -2.004062 | 0.0000000 |
| Taf1d | -0.7711545 | 0.0000000 | -1.291331 | 0.0000000 |
| Tipin | -0.6868983 | 0.0000000 | -1.924255 | 0.0000001 |
| Ireb2 | -0.5745806 | 0.0000000 | -1.577767 | 0.0000000 |
| Dbr1 | -1.2502667 | 0.0000000 | -1.068616 | 0.0000000 |
| Herc1 | -0.8480354 | 0.0000000 | -3.063395 | 0.0000000 |
| Nktr | -1.6595711 | 0.0000000 | -3.278782 | 0.0000000 |
| Qrich1 | -0.6928870 | 0.0000000 | -1.025579 | 0.0000000 |
| Scamp5 | -1.2507434 | 0.0000000 | -2.102386 | 0.0000000 |
| Glce | -1.0766042 | 0.0000000 | -1.431818 | 0.0000000 |
| Wdr6 | -0.5528243 | 0.0000000 | -1.442052 | 0.0000000 |
| Map2k2 | -0.6466754 | 0.0000000 | -1.869505 | 0.0000000 |
| Tex9 | -1.3196501 | 0.0000000 | -2.494112 | 0.0000000 |
| Rsph4a | -0.6344661 | 0.0000207 | -1.272442 | 0.0051859 |
| Sec63 | -0.8516400 | 0.0000000 | -1.317850 | 0.0000000 |
| Dctn2 | -0.5404288 | 0.0000000 | -1.124960 | 0.0001331 |
| Ppp1r12a | -0.6554683 | 0.0000000 | -1.111509 | 0.0000000 |
| Rnf167 | -1.1745182 | 0.0000000 | -1.765360 | 0.0000000 |
| Nprl3 | -1.5239045 | 0.0000000 | -2.352325 | 0.0000057 |
| Spdl1 | -1.6981270 | 0.0000000 | -1.974112 | 0.0000000 |
| Cdk12 | -1.7089477 | 0.0000000 | -3.606867 | 0.0000000 |
| Zfp867 | -1.5832032 | 0.0000000 | -1.750745 | 0.0000000 |
| Gm17586 | -1.0264258 | 0.0000000 | -1.694781 | 0.0000126 |
| Haspin | -1.6247701 | 0.0000000 | -1.730556 | 0.0000065 |
| Cyb5d1 | -1.3738235 | 0.0000000 | -1.978572 | 0.0000002 |
| Cops3 | -0.1625868 | 0.0592244 | -1.006107 | 0.0000000 |
| Camk2b | -0.2123757 | 0.0452834 | -1.096598 | 0.0262078 |
| Cox11 | -1.0678872 | 0.0000000 | -1.614065 | 0.0000003 |
| Tmem18 | -2.0734860 | 0.0000000 | -3.274443 | 0.0000000 |
| Ankfy1 | -0.9841575 | 0.0000000 | -2.020487 | 0.0000000 |
| Drg1 | -0.3494618 | 0.0000012 | -1.472060 | 0.0000000 |
| 4930447C04Rik | -0.7537949 | 0.0000000 | -2.659638 | 0.0000092 |
| Dnajc27 | -2.1419602 | 0.0000000 | -3.400425 | 0.0000000 |
| Churc1 | -0.6628013 | 0.0000000 | -2.641795 | 0.0000000 |
| Stxbp4 | -0.6779614 | 0.0000000 | -1.980874 | 0.0000698 |
| Med1 | -2.5015464 | 0.0000000 | -3.575061 | 0.0000000 |
| Taok1 | -1.2255178 | 0.0000000 | -1.961266 | 0.0000000 |
| Zfp386 | -1.0345934 | 0.0000000 | -1.348782 | 0.0000000 |
| Sipa1l1 | -0.5652762 | 0.0000000 | -2.355421 | 0.0000000 |
| Ercc6l2 | -0.6195863 | 0.0000000 | -3.898004 | 0.0000000 |
| Zfp346 | -1.2157077 | 0.0000000 | -1.600378 | 0.0000201 |
| Cab39l | -0.3807831 | 0.0000005 | -2.133212 | 0.0000112 |
| Micu2 | -0.1761896 | 0.3451150 | -1.123774 | 0.0061334 |
| Camk2g | -1.4873715 | 0.0000000 | -2.057711 | 0.0000000 |
| Usp54 | -0.7536751 | 0.0000000 | -1.320395 | 0.0000000 |
| Akap11 | -0.6919425 | 0.0000000 | -1.679484 | 0.0000000 |
| Dis3 | -0.3998322 | 0.0000000 | -1.003921 | 0.0000000 |
| Cyc1 | -0.2758691 | 0.0000745 | -1.246631 | 0.0001025 |
| Puf60 | -0.5987206 | 0.0000000 | -1.612616 | 0.0000000 |
| Mkl2 | -1.3637457 | 0.0000000 | -2.273677 | 0.0000000 |
| Gm28047 | -1.2548423 | 0.0000000 | -1.787430 | 0.0096741 |
| Atf7 | -1.5128512 | 0.0000000 | -2.258619 | 0.0000000 |
| Arf3 | -0.9204991 | 0.0000000 | -3.785531 | 0.0000000 |
| Dzip3 | -0.5710258 | 0.0000000 | -2.319581 | 0.0000017 |
| Nsun3 | -0.9790886 | 0.0000000 | -1.543068 | 0.0080199 |
| Zfp414 | -1.1357843 | 0.0000000 | -1.852831 | 0.0000017 |
| B230354K17Rik | -1.1794817 | 0.0000000 | -1.387088 | 0.0000000 |
| Chaf1a | -1.2071751 | 0.0000000 | -1.388689 | 0.0000000 |
| Gm6741 | -0.7776053 | 0.0000000 | -1.006212 | 0.0015078 |
| Mapk14 | -1.5844714 | 0.0000000 | -3.457840 | 0.0000006 |
| Rpl7a-ps5 | -0.1272856 | 0.2705847 | -1.056035 | 0.0239379 |
| Cd2ap | -0.5476119 | 0.0000000 | -1.172565 | 0.0000000 |
| Eif2ak2 | -0.8770576 | 0.0000000 | -2.086080 | 0.0000123 |
| Polr1c | -1.0626646 | 0.0000000 | -1.666996 | 0.0000000 |
| Myo5b | -0.2075730 | 0.1107000 | -1.307604 | 0.0132828 |
| Rpl17 | -0.2429525 | 0.0064102 | -1.270315 | 0.0000147 |
| Dmxl1 | -0.7864044 | 0.0000000 | -1.180367 | 0.0000000 |
| Maoa | -0.2142286 | 0.0262770 | -1.357778 | 0.0033018 |
| Uhrf2 | -1.2972704 | 0.0000000 | -2.727566 | 0.0000000 |
| Wdr44 | -0.9717873 | 0.0000000 | -1.178816 | 0.0001895 |
| Hmgb3 | -0.3387660 | 0.0051868 | -1.350774 | 0.0341549 |
| Zmym3 | -0.3710260 | 0.0000000 | -1.242310 | 0.0000001 |
| Trappc2 | -0.6661864 | 0.0000000 | -1.859098 | 0.0056808 |
| Hells | -0.7293904 | 0.0000000 | -2.054234 | 0.0000000 |
| Cox7b | -0.2073749 | 0.1992399 | -1.245849 | 0.0110895 |
| Fundc2 | -0.3890425 | 0.0002625 | -1.422037 | 0.0070256 |
| Nkrf | -1.8445290 | 0.0000000 | -2.183910 | 0.0000000 |
| Ofd1 | -0.6918110 | 0.0000000 | -2.349520 | 0.0000000 |
| Idh3g | -0.2804031 | 0.0002062 | -1.358422 | 0.0001946 |