DESeq2: maxCooks not set (NA)
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Entering edit mode
ipso • 0
@ipso-9761
Last seen 4.3 years ago

Hi,

I've got a question regarding the Cooks distance calculation in DESeq2. 
My dataset consists of 4 groups with 5-6 biological replicates per group. For some exploratory QC plotting I performed the GLM fitting with different design matrices, one with the batch as covariate (dds.bc) and one with only my group of interest (dds.nbc):

# Read summarized experiment
se <- readRDS(se.path)

# Prepare colData
group = factor(se$group)
batch = factor(se$exp)
col.data <- data.frame(row.names=colnames(se), group, batch)

# Create dds with batch correction
dds.bc <- DESeq2::DESeqDataSetFromMatrix(countData = assay(se, "counts"),
                                         colData = col.data,
                                         design = ~batch+group)

# Create dds without batch correction
dds.nbc <- DESeq2::DESeqDataSetFromMatrix(countData = assay(se, "counts"),
                                          colData = col.data,
                                          design = ~group)

# Calculate differential expression
dds.bc <- DESeq(dds.bc, betaPrior = FALSE, fitType = 'local')
dds.nbc <- DESeq(dds.nbc, betaPrior = FALSE, fitType = 'local')

Now for some reason the maxCooks value (rowData(dds)['maxCooks']) is only set for the analysis using only the group in the design formula (dds.nbc). In dds.bc the values are all set to 'NA':

> head(rowData(dds.bc)['maxCooks'])
DataFrame with 6 rows and 1 column
   maxCooks
  <logical>
1        NA
2        NA
3        NA
4        NA
5        NA
6        NA
> head(rowData(dds.nbc)['maxCooks'])
DataFrame with 6 rows and 1 column
           maxCooks
          <numeric>
1   2.5255207003168
2 0.657178470968108
3  1.27111489329578
4 0.144701162255673
5  2.65777960268025
6  13.1324009789259​

The Cooks distances are calculated (assay(dds, 'cooks')) but not transferred to the column in the rowData:

> head(assay(dds.nbc, 'cooks'))

                     a1        a2         a3        a4        a5         a6      b1      b2      b3     b4      b5
0610007P14Rik 0.056467175 0.032335019 1.441970e+00 0.057708134 0.058649497 0.0577954102 0.043284253 0.063377144 0.014263303 0.09582077 1.806441441
0610009L18Rik 0.657178471 0.018659529 3.290204e-02 0.032453661 0.033013610 0.0325055582 0.006252503 0.009041238 0.013313577 0.01367133 0.013652200
0610009O20Rik 0.076308209 0.066229282 1.133886e-02 0.076655314 1.271114893 0.0363556309 0.005891674 0.008305582 0.011777309 0.01205639 0.012041508
0610010B08Rik 0.004603687 0.003851429 2.935343e-02 0.004630839 0.004650915 0.0046327188 0.003165467 0.003751235 0.004327378 0.00436450 0.004362548
0610010F05Rik 0.066643541 0.108785212 5.065448e-05 0.011146832 0.024336550 0.0005165392 0.101602398 2.657779603 0.119600118 0.12444698 0.124415077
0610010K14Rik 0.097618110 0.027128868 3.158805e-01 0.049495746 0.050334776 0.0495735232 0.006252688 0.009041506 0.013313970 0.01367173 0.013652602
                   b6       c1       c2       c3       c4       c5       c6      d1      d2       d3
0610007P14Rik 0.086250911 7.766767e-03 7.774940e-03 1.349925e-02 1.373434e-02 1.178278e-02 1.309203e-02 0.059908254 0.145012806 2.5255207003
0610009L18Rik 0.012288280 7.766634e-03 7.774807e-03 1.349902e-02 1.373411e-02 1.178258e-02 1.309180e-02 0.020024088 0.048928772 0.5657196744
0610009O20Rik 0.010967809 9.780299e-03 9.789584e-03 1.585565e-02 1.608741e-02 1.412400e-02 7.858235e-02 0.045306524 0.075009889 1.2260605501
0610010B08Rik 0.004213122 5.358793e-03 5.360122e-03 5.938908e-03 1.447012e-01 5.817130e-03 5.912585e-03 0.007867464 0.008202417 0.0009390616
0610010F05Rik 0.088157912 2.292114e-07 2.294538e-07 3.999231e-07 4.069522e-07 3.486694e-07 3.877527e-07 0.046491910 0.079683380 1.2482338185
0610010K14Rik 0.012288643 7.766659e-03 7.774831e-03 1.349906e-02 1.373415e-02 1.178262e-02 1.309184e-02 0.492206223 0.880840891 0.9371058211
                   d4      d5
0610007P14Rik  0.14581105 0.101974129
0610009L18Rik  0.04922949 0.033502302
0610009O20Rik  0.07519935 0.062800918
0610010B08Rik  0.05747505 0.008097566
0610010F05Rik  0.07991326 0.065384214
0610010K14Rik 13.13240098 0.525006091

Is there a reason for this that I am missing? Is it okay to just manually add the values?

rowData(dds.bc)['maxCooks'] <- apply(assay(dds.bc, 'cooks'), 1, max)​

Also, when only using the group in the design formula, the analysis is detecting 20% outliers. Would you object to setting the minReplicatesForReplace to 5 or 6 to deal with this?

Thank you very much,
Joe

> sessionInfo()
R version 3.5.1 (2018-07-02)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS High Sierra 10.13.6

Matrix products: default
BLAS: /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib

locale:
[1] de_DE.UTF-8/de_DE.UTF-8/de_DE.UTF-8/C/de_DE.UTF-8/de_DE.UTF-8

attached base packages:
[1] parallel  stats4    stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] DESeq2_1.20.0               SummarizedExperiment_1.10.1 DelayedArray_0.6.5          BiocParallel_1.14.2         matrixStats_0.54.0         
 [6] Biobase_2.40.0              GenomicRanges_1.32.6        GenomeInfoDb_1.16.0         IRanges_2.14.10             S4Vectors_0.18.3           
[11] BiocGenerics_0.26.0  
deseq2 cooks distance dds • 773 views
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Entering edit mode
@mikelove
Last seen 4 days ago
United States

From ?results:

Note: this test excludes the Cook's distance of samples belonging to experimental groups with only 2 samples.

In order to filter based on Cook's you need 3 or more samples in the same "cell", where by "cell" I mean unique combination of all the factors in the design. So when you add batch, you break up the cells so that there are no longer 3 or more samples.

I would take a look by eye at some of the genes with outliers (using plotCounts) and then if you feel confident that these outliers would be better replaced then you could set minReplicatesForReplace to a lower value.

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Entering edit mode

Thank you Michael, I obviously missed that note! And thank you also for the advice regarding the outlier replacement! :)

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