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)
})<- ReadGRAND("https://zenodo.org/record/6976391/files/BANP.tsv.gz",
data 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)
<- FilterGenes(data) 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
<- GetContrasts(data,contrast=c("Experimental.time.original","0h"),columns = Genotype=='dTag')
contrasts <- 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") data
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):
<- GetSignificantGenes(data,analysis = "new.4h vs 0h", criteria = Q<0.05 & LFC< -1)
genes 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 |