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Annotate ouput file from Deseq2
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18 months ago
BM • 40
United Kingdom

I am trying to annotate the results output file from Desq2 so it contains gene names and symbols. The RNA-seq count file I have used comes from Dexseq and contains ensembl transcript ID:

ENSMUSG00000000001:001

ENSMUSG00000000001:002

ENSMUSG00000000001:003

etc.

I have tried various methods to annotate the results.

1. downloaded annotation from Biomart.

library(DESeq2)

counts = read.delim("3mTA2.txt", header=T, row.names=1)

sample <- read.delim("~/sample.txt")

count.data.set <- DESeqDataSetFromMatrix(countData=counts, colData=sample,design= ~ genotype)

dds<-DESeq(count.data.set)

res <- results(dds)

annotation <- read.delim("mouse.annt.txt") # load annotation file from Biomart

res$EnsemblID <- row.names(res) res <- merge(res, annotation, by = 'EnsemblID', all.x = TRUE) It adds column to the output file but values are blank. 2. Also used AnnotationDbi library("AnnotationDbi") library("org.Mmu.eg.db") res$symbol <- mapIds(org.Mmu.eg.db,

• keys=row.names(res),

• column="SYMBOL",

• keytype="ENSEMBL",

• multiVals="first")

Error in .testForValidKeys(x, keys, keytype) :

None of the keys entered are valid keys for 'ENSEMBL'. Please use the keys method to see a listing of valid arguments.

Any sugestions?

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15 months ago
James Ashmore ♦ 2.6k
UK/Edinburgh/MRC Centre for Regenerativ…

Those IDs you have listed are Ensembl gene IDs, not transcript IDs. I'm also not sure why they have the ':001' string after them? If you try the BioMart id conversion tool you can see that if you remove this last part and convert the ID to a gene name you get a result e.g. ENSMUSG00000000001 = GNAI3. This Ensembl tutorial may help you discern between the different IDs.

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ENSMUSG00000000001:001; ENSMUSG00000000001:002 - these refer to the the different exons of the gene.

So the question I suppose is how to combine or merge the different exon counts for the same gene into one count for the gene?

Can this be done in Dexseq or Deseq2?

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You don't want to do that, since doing so will double count a number of things. Just run either htseq-count or featureCounts (this is much faster) and directly get gene level metrics.

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The initial analysis was performed elsewhere. So I only have the Dexseq count file with ensemble ids of all the different exons of a gene. How can i use this file to proceed - either by annotating exons ids into a gene or using the file in Deseq2 and then annotate ?

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That's unfortunate, particularly if you don't have the BAM or fastq files. Indeed, the best you can do is just remove the :E??? from the names, sum over the results and use that. Note that the results will then be approximate. You could do that with awk.

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