Post detailing the Multi-Species results from DESeq2 –> DEG lists and annotations.

Summary

This post links out all the key results files as of now for differential expression analyses. The count matrices for each species, the DESeq2 results for all the different comparisons, and the annotated DEG lists from those differential gene expression analyses.

Some overall notes:

all results are based off of the transcript matrices for each species
the different DESeq2 analyses within each species section are as follows:

Combing time points (day 6 and 12) and just comparing exposed to control within each species: Design = ~ condition (SSWD-exposed vs control)
Design = ~ day + condition –> comparing condition (SSWD-exposed or not) while controlling for day (day 6 or 12)
Contrast on the comparison from 2 above –> is the same as the DEG list in 4 below: compares time points (day 6 vs day 12) while controlling for condition (SSWD-exposed vs not). From the DESeq2 manual: “A contrast is a linear combination of estimated log2 fold changes. Can be used to test if differences between groups are equal to zero.” To get the contrast, you do: deseq2.resday <- results(ddsDay, contrast = c("day", "6", "12")), which, again, is the SAME THING as Design = ~ condition + day below.
Design = ~ condition + day –> a different way to get the same results as 3 above –> comparing time points (day 6 vs day 12) while controlling for condition (SSWD-exposed or not)
Design = ~ condition + day + condition:day –> interaction. Looks to see if the effect of SSWD-exposure depends on day (6 vs 12) by adding an interaction term (condition:day). Both condition (SSWD-exposure or not) and day (6 vs 12) are main effects, and to see if they depend on each other to result in a gene expression outcome, you need the interaction term of the two (condition:day).
Comparing control to exposed from JUST day 6: Design = ~ condition (JUST for the libraries from day 6 – comparing SSWD-exposed to control)
Comparing control to exposed from JUST day 12: Design = ~ condition (JUST for the libraries from day 12 – comparing SSWD-exposed to control)

Key Links:

Project Repository:

project-pycno-multispecies-2023

(NOTE: repository is PRIVATE until collaborators publish their genomic data - links in this notebook post will ONLY work for current collaborators)

Count Matrices:

`DESeq2` Results:

P. helianthoides

All results are from transcript count matrices

DESeq2
Code: project-pycno-multispecies-2023/code/19-deseq2-pycno.Rmd

DEGs combining Day 6 and Day 12: project-pycno-multispecies-2023/output/19-deseq2-pycno/DEGlist_transcripts_pycno_controlVexposed.tab
Design = ~ condition
1893 DEGs (<0.05 padj)

DEGs controlling for time points (Day 6 vs Day 12) and comparing condition (SSWD exposure or not): project-pycno-multispecies-2023/output/19-deseq2-pycno/DEGlist_transcripts_pycno_CvEwithDay.tab
Design = ~ day + condition
2252 DEGs (<0.05 padj)

summary(ddsDay.res)
out of 23353 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 1930, 8.3%
LFC < 0 (down)     : 1367, 5.9%
outliers [1]       : 439, 1.9%
low counts [2]     : 5970, 26%
(mean count < 15)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

Contrast on day from above:
Now need to perform a contrast to see if there is a difference between these groups as it relates to day project-pycno-multispecies-2023/output/19-deseq2-pycno/DEGlist_transcripts_pycno_CvEwithDay_contrast.tab
791 DEGs (<0.05 padj)
To get the contrast:
```
deseq2.resday <- results(ddsDay,
                       contrast = c("day", "6",  "12"))
```

Files 3 and 4 are the exact same… just two different methods of getting the same file.

DEGs comparing time points (Day 6 vs Day 12) and controlling for condition (SSWD-exposure or not):
project-pycno-multispecies-2023/output/19-deseq2-pycno/DEGlist_transcripts_pycno_controlforcondition-compare-timepoints.tab
Design = ~ condition + day
791 DEGs (<0.05 padj)

summary(ddsDayCon.res)
out of 23353 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 723, 3.1%
LFC < 0 (down)     : 840, 3.6%
outliers [1]       : 439, 1.9%
low counts [2]     : 7294, 31%
(mean count < 30)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

DEGs with an interaction term –> seeing if effects of SSWD-exposure and/or time change over time:
project-pycno-multispecies-2023/output/19-deseq2-pycno/DEGlist_transcripts_pycno_interaction.tab
Design = ~ condition + day + condition:day
4003 DEGs (<0.05 padj)

summary(ddsDayInteract.res)
out of 23353 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 2641, 11%
LFC < 0 (down)     : 2539, 11%
outliers [1]       : 268, 1.1%
low counts [2]     : 4793, 21%
(mean count < 7)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

DEGs Day 6 alone - exposed vs control : project-pycno-multispecies-2023/output/19-deseq2-pycno/DEGlist_transcripts_pycno_controlVexposed_DAY6.tab
Design = ~ condition
109 DEGs (<0.05 padj)
DEGs Day 12 alone - exposed vs control: project-pycno-multispecies-2023/output/19-deseq2-pycno/DEGlist_transcripts_pycno_controlVexposed_DAY12.tab
Design = ~ condition
5359 DEGs (<0.05 padj)

Annotating
Code: project-pycno-multispecies-2023/code/22-annot-DEGlists.Rmd

Annotated lists correspond to the seven DEGlists listed above for Pycnopodia helianthoides

P. ochraceus

DESeq2
Code: project-pycno-multispecies-2023/code/20-deseq2-pisaster.Rmd

DEGs combining Day 6 and Day 12: project-pycno-multispecies-2023/output/20-deseq2-pisaster/DEGlist_pisaster_controlVexposed.tab
Design = ~ condition
2262 DEGs (<0.05 padj)

DEGs controlling for time points (Day 6 vs Day 12) and comparing condition (SSWD exposure or not): project-pycno-multispecies-2023/output/20-deseq2-pisaster/DEGlist_transcripts_pisaster_CvEwithDay.tab
Design = ~ day + condition
3054 DEGs (<0.05 padj)

summary(ddsDay.res)
out of 31263 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 3491, 11%
LFC < 0 (down)     : 1197, 3.8%
outliers [1]       : 1702, 5.4%
low counts [2]     : 6590, 21%
(mean count < 2)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

Contrast on day from above: project-pycno-multispecies-2023/output/20-deseq2-pisaster/DEGlist_transcripts_pisaster_CvEwithDay_contrast.tab
333 DEGs (<0.05 padj)
To get the contrast:
```
deseq2.resday <- results(ddsDay,
                       contrast = c("day", "6",  "12"))
```

Files 3 and 4 are the exact same… just two different methods of getting the same file.

DEGs comparing time points (Day 6 vs Day 12) and controlling for condition (SSWD-exposed or not): project-pycno-multispecies-2023/output/20-deseq2-pisaster/DEGlist_transcripts_pisaster_controlforcondition-compare-timepoints.tab
Design = ~ condition + day
333 DEGs (<0.05 padj)

summary(ddsDayCon.res)
out of 31263 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 256, 0.82%
LFC < 0 (down)     : 493, 1.6%
outliers [1]       : 1702, 5.4%
low counts [2]     : 10905, 35%
(mean count < 13)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

DEGs with an interaction term –> seeing if effects of SSWD-exposure and/or time change over time: project-pycno-multispecies-2023/output/20-deseq2-pisaster/DEGlist_transcripts_pisaster_interaction.tab
Design = ~ condition + day + condition:day
818 DEGs (<0.05 padj)

summary(ddsDayInteract.res)
out of 31263 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 311, 0.99%
LFC < 0 (down)     : 931, 3%
outliers [1]       : 1227, 3.9%
low counts [2]     : 8503, 27%
(mean count < 4)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

DEGs Day 6 alone - exposed vs control: project-pycno-multispecies-2023/output/20-deseq2-pisaster/DEGlist_transcripts_pisaster_controlVexposed_DAY6.tab
Design = ~ condition
80 DEGs (<0.05 padj)
DEGs Day 12 alone - exposed vs control: project-pycno-multispecies-2023/output/20-deseq2-pisaster/DEGlist_transcripts_pisaster_controlVexposed_DAY12.tab
Design = ~ condition
4558 DEGs (<0.05 padj)

Annotating
Code: project-pycno-multispecies-2023/code/22-annot-DEGlists.Rmd

Annotated lists correspond to the seven DEGlists listed above for Pisaster ochraceus

D. imbricata

DESeq2
Code: project-pycno-multispecies-2023/code/21-deseq2-derm.Rmd

DEGs combining Day 6 and Day 12: project-pycno-multispecies-2023/output/21-deseq2-derm/DEGlist_derm_controlVexposed.tab
Design = ~ condition
375 DEGs (<0.05 padj)

DEGs controlling for time points (Day 6 vs Day 12) and comparing condition (SSWD exposure or not): project-pycno-multispecies-2023/output/21-deseq2-derm/DEGlist_transcripts_derm_CvEwithDay.tab
Design = ~ day + condition
708 DEGs (<0.05 padj)

summary(ddsDay.res)
out of 23164 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 675, 2.9%
LFC < 0 (down)     : 640, 2.8%
outliers [1]       : 1059, 4.6%
low counts [2]     : 3580, 15%
(mean count < 6)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

Contrast on day from above: project-pycno-multispecies-2023/output/21-deseq2-derm/DEGlist_transcripts_derm_CvEwithDay_contrast.tab
1775 DEGs (<0.05 padj)
To get the contrast:
```
deseq2.resday <- results(ddsDay,
                       contrast = c("day", "6",  "12"))
```

Files 3 and 4 are the exact same… just two different methods of getting the same file.

DEGs comparing time points (Day 6 vs Day 12) and controlling for condition (SSWD-exposed or not): project-pycno-multispecies-2023/output/21-deseq2-derm/DEGlist_transcripts_derm_controlforcondition-compare-timepoints.tab
Design = ~ condition + day
1775 DEGs (<0.05 padj)

summary(ddsDayCon.res)
out of 23164 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 1695, 7.3%
LFC < 0 (down)     : 1138, 4.9%
outliers [1]       : 1059, 4.6%
low counts [2]     : 6002, 26%
(mean count < 34)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

DEGs with an interaction term –> seeing if effects of SSWD-exposure and/or time change over time: project-pycno-multispecies-2023/output/21-deseq2-derm/DEGlist_transcripts_derm_interaction.tab
Design = ~ condition + day + condition:day
547 DEGs (<0.05 padj)

summary(ddsInteract.res)
out of 23164 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up)       : 321, 1.4%
LFC < 0 (down)     : 424, 1.8%
outliers [1]       : 806, 3.5%
low counts [2]     : 3744, 16%
(mean count < 6)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

DEGs Day 6 alone - exposed vs control: project-pycno-multispecies-2023/output/21-deseq2-derm/DEGlist_transcripts_derm_controlVexposed_DAY6.tab
Design = ~ condition
38 DEGs (<0.05 padj)
DEGs Day 12 alone - exposed vs control: project-pycno-multispecies-2023/output/21-deseq2-derm/DEGlist_transcripts_derm_controlVexposed_DAY12.ta
Design = ~condition
2787 DEGs (<0.05 padj)

Annotating
Code: project-pycno-multispecies-2023/code/22-annot-DEGlists.Rmd

Annotated lists correspond to the seven DEGlists listed above for Dermasterias imbricata

Summary

Some overall notes:

Key Links:

Project Repository:

Count Matrices:

P. helianthoides

Pisaster ochraceus

Dermasterias imbricata

DESeq2 Results:

P. helianthoides

All results are from transcript count matrices

P. ochraceus

D. imbricata

`DESeq2` Results: