Differential gene expression between conditions from bulk RNA-seq data.
Input
Paired-end RNA-seq reads from samples in two or more conditions.
Output
The log2 fold change and its significance for every gene, for each comparison between conditions.
Validation
We analysed the publicly available RNA-seq data of the reference study, which compared dexamethasone-treated with untreated primary human airway smooth muscle cells from four donors, and compared each of the 28 results reported in that study with the corresponding result of this reproduction. All 28 results were reproduced (Figure 1). The log2 fold changes of the 17 genes in Table 1 of the reference study agreed closely with the reported values (Pearson r = 0.9988; Figure 2). The number of differentially expressed genes (q < 0.05) was 300, compared with 316 reported, and 278 of the 316 reported genes (88.0%) were differentially expressed in the same direction in this reproduction (Figure 3). The mapping rate (%) of every sample was within or above the reported range (Figure 4).
Figure 1. Reproduction of results reported in the reference study (n = 28)
Show data table
Reported result
Outcome
Values
Number of differentially expressed genes (q < 0.05)
Reproduced
300 versus 316 reported
C7 log2 fold change
Reproduced
CCDC69 log2 fold change
Reproduced
DUSP1 log2 fold change
Reproduced
FKBP5 log2 fold change
Reproduced
GPX3 log2 fold change
Reproduced
KLF15 log2 fold change
Reproduced
MAOA log2 fold change
Reproduced
SAMHD1 log2 fold change
Reproduced
SERPINA3 log2 fold change
Reproduced
SPARCL1 log2 fold change
Reproduced
C13orf15 log2 fold change
Reproduced
TSC22D3 log2 fold change
Reproduced
CRISPLD2 log2 fold change
Reproduced
PER1 log2 fold change
Reproduced
KCTD12 log2 fold change
Reproduced
ERRFI1 log2 fold change
Reproduced
STEAP4 log2 fold change
Reproduced
Overlap with the reported differentially expressed genes
Reproduced
CEBPD: differentially expressed, increased
Reproduced
CEBPB: expressed, not differentially expressed
Reproduced
SYNPO2: differentially expressed
Reproduced
B2M: highly expressed, not differentially expressed
Reproduced
GABARAP: highly expressed, not differentially expressed
Reproduced
GAPDH: highly expressed, not differentially expressed
Reproduced
RPL19: highly expressed, not differentially expressed
Reproduced
IL1B: low expression in both conditions
Reproduced
Mean mapping rate (%)
Reproduced
Each symbol is one result reported in the reference study: the log2 fold changes of the 17 genes in Table 1, the overlap with the reported list of differentially expressed genes, eight statements on the expression of individual genes, the mean mapping rate and the number of differentially expressed genes. Filled circles denote results reproduced in this analysis. All 28 results were reproduced. The number of differentially expressed genes (q < 0.05) was 300, compared with 316 reported.
Figure 2. Concordance of log2 fold changes for the 17 genes in Table 1 of the reference study
Show data table
Gene
Reported
Reproduced
Difference
C7
3.35
3.21
−0.14
CCDC69
2.92
2.87
−0.05
DUSP1
2.99
2.95
−0.04
FKBP5
3.95
3.92
−0.03
GPX3
3.76
3.72
−0.04
KLF15
4.58
4.57
−0.01
MAOA
3.29
3.3
0.01
SAMHD1
3.83
3.84
0.01
SERPINA3
3.34
3.28
−0.06
SPARCL1
4.7
4.6
−0.1
C13orf15
3.27
3.17
−0.1
TSC22D3
3.27
3.24
−0.03
CRISPLD2
2.7
2.69
−0.01
PER1
3.2
3.19
−0.01
KCTD12
−2.52
−2.49
0.03
ERRFI1
2.43
2.44
0.01
STEAP4
4.99
5.22
0.23
Each point is one gene; the horizontal axis gives the log2 fold change reported in the reference study and the vertical axis the log2 fold change in this reproduction (dexamethasone-treated versus untreated; n = 4 donors). The diagonal marks identity (y = x). All 17 estimates fall within 0.24 log2 units of the reported values (mean absolute difference 0.054; Pearson r = 0.9988). The largest difference is for STEAP4 (5.225 versus 4.99 reported).
Figure 3. Reproduction of the 316 genes reported as differentially expressed (q < 0.05)
Show data table
Category
Genes
Percentage
reproduced (same direction)
278
88.0%
not reproduced
38
12.0%
Total
316
100%
The bar represents the 316 genes reported as differentially expressed (q < 0.05) in the reference study (Table S3), divided according to whether each gene is also differentially expressed in this reproduction with the same direction of change. Of the 316 genes, 278 (88.0%) were reproduced and 38 (12.0%) were not.
Figure 4. Mapping rate per sample relative to the reported range
Show data table
Sample
Value (%)
SRR1039508
85.9%
SRR1039509
84.6%
SRR1039512
85.9%
SRR1039513
85.5%
SRR1039516
84%
SRR1039517
85.2%
SRR1039520
85.5%
SRR1039521
85.8%
Reported range
81.9% to 84.3%
Reported mean
83.4%
Each point is one sample (n = 8; four donors, treated and untreated). The shaded band marks the per-sample range reported in the reference study (81.9–84.3%) and the vertical line the reported mean (83.4%). The mean mapping rate in this reproduction is 85.3% (range 84.0–85.9%). Of the 8 samples, 7 lie above the reported range, 1 within it and 0 below it.
Reference
Himes BE, Jiang X, Wagner P, et al. RNA-Seq transcriptome profiling identifies CRISPLD2 as a glucocorticoid responsive gene that modulates cytokine function in airway smooth muscle cells. PLoS One. 2014;9(6):e99625. doi:10.1371/journal.pone.0099625