hi Jie,

The warning continues in DESeq2, saying that a local fit was substituted
for the parametric fit, and to check the plot of dispersion estimates using
plotDispEsts() for quality of the local regression curve. I should probably
extend the text of the warning, because users seem to get stuck here
thinking that they cannot continue. It means that the software tried the
default method, but could not fit the parametric curve through the
dispersion estimates over the mean counts. The parametric function works
well for most but not all RNA-Seq experiments. You should then follow the
instructions of the next line of the warning and check the plot to make
sure that the local regression curve is not being overly influenced by
outlier points. If it seems a good fit, then you can continue with the
analysis as is.  if not, then you should instead use fitType="mean" with
DESeq().

Mike



On Mon, Nov 25, 2013 at 9:52 AM, Jiewencai <jiewencai@qq.com> wrote:

> Hi,
>
>
> I have 4 sample of RNA-Seq read counts data, The data are like below:
>
>
> gene_id CK-1    CK-2    T-1     T-2
> isoform1        66      78      133     150
> isoform2        2106    1764    1965    1894
> isoform3        17452   19469   21509   27042
> isoform4        156     165     7       9
>
>
> where CK-1 and CK-2 are replicates of the one condition,
> T-1 and T-2 are replicates of the other condition.
>
>
> When I try to find differential expression isoforms by DESeq or DESeq2,
> I got the Warning: "Dispersion fit did not converge".
>
>
> Simon said when analysis data without replicates would arise this warning.
> But my data do  have replicates, Why this warning occurred ? Is there any
> problems
> with my count data ? Would this warning reduce my result's accuracy ?
>
>
> Any help is appreciated !
>
>
> Best,
> Wencai Jie
>
>
> ###########################  DESeq commands
>  #######################################
> library("DESeq")
> condition = factor(c("CK","CK","Treated","Treated"))
> countTable = read.table("counts.txt",header=T,row.name=1)
> cds = newCountDataSet(countTable,condition)
> cds  =  estimateSizeFactors(cds)
> cds  =  estimateDispersions(cds)
>
>
> Warning:
> In parametricDispersionFit(means, disps) : Dispersion fit did not converge.
>
>
> ###########################  DESeq2 commands
>  #######################################
> library("DESeq2")
> countData = read.table("counts.txt",header=T,row.names=1)
> colData = DataFrame(condition=factor(c("CK","CK","Treated","Treated")))
> dds = DESeqDataSetFromMatrix(countData=countData,colData=colData,design =~
> condition)
> dds = DESeq(dds)
>
>
> Warning:
> In parametricDispersionFit(mcols(objectNZ)$baseMean[useForFit],  :
>   dispersion fit did not converge
>
>
>
>
>
> ####################################################################################
> sessionInfo()
>
>
> R version 3.0.1 (2013-05-16)
> Platform: x86_64-w64-mingw32/x64 (64-bit)
>
>
> locale:
> [1] LC_COLLATE=Chinese (Simplified)_China.936
> [2] LC_CTYPE=Chinese (Simplified)_China.936
> [3] LC_MONETARY=Chinese (Simplified)_China.936
> [4] LC_NUMERIC=C
> [5] LC_TIME=Chinese (Simplified)_China.936
>
>
> attached base packages:
> [1] parallel  stats     graphics  grDevices utils     datasets  methods
> [8] base
>
>
> other attached packages:
> [1] DESeq2_1.0.19           RcppArmadillo_0.3.920.3 Rcpp_0.10.6
> [4] lattice_0.20-24         Biobase_2.20.1          GenomicRanges_1.12.5
> [7] IRanges_1.18.4          BiocGenerics_0.6.0
>
>
> loaded via a namespace (and not attached):
>  [1] annotate_1.38.0      AnnotationDbi_1.22.6 DBI_0.2-7
>  [4] genefilter_1.42.0    grid_3.0.1           locfit_1.5-9.1
>  [7] RColorBrewer_1.0-5   RSQLite_0.11.4       splines_3.0.1
> [10] stats4_3.0.1         survival_2.37-4      XML_3.98-1.1
> [13] xtable_1.7-1
>         [[alternative HTML version deleted]]
>
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