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Old 07-28-2015, 08:08 AM   #1
imsharmanitin
Postdoc Cancer Bioinformatics
 
Location: Olso, Norway

Join Date: Dec 2014
Posts: 17
Cool Differential expression using TopHat/Cufflinks, analysis by CumeRbund

I am trying to do differential expression analysis for 6 samples with no replicates

The protocol used by me after following several threads and http://www.nature.com/nprot/journal/....2012.016.html
is as follows

1) after steps like alignment, etc

2) I assembled transcripts for each sample
cufflinks -p 8 -o <output directory> accepted_hits.bam

5) Ran Cuffmerge to create a single merged transcriptome annotation:
cuffmerge -g Homo_sapiens.GRCh37.75.gtf -s Homo_sapiens.GRCh37.75.dna.primary_assembly.fa -p 8 assemblies.txt

6) ran cuffdiff using following options:
--library-type fr-unstranded
--dispersion-method blind -L C1,C2,C3,C4,C5,C6
-b ./index/Homo_sapiens.GRCh37.75.dna.primary_assembly.fa
-u ./merged_asm/merged.gtf

7) used CumeRbund for analysis


However, I was unable to get Ensemble id and instead got XLOC id for the genes.


following the thread http://seqanswers.com/forums/showthread.php?t=18357
I tried following two options

1) featureNames(sigGenes)

tracking_id gene_short_name
1 XLOC_002638 HES4,RP11-54O7.17
2 XLOC_005270 <NA>
3 XLOC_005288 <NA>
4 XLOC_005368 <NA>
5 XLOC_007367 RP11-47A8.5
6 XLOC_007664 <NA>
7 XLOC_007703 <NA>

But the names for some of XLOC were missing

2) then i tired other method and i got error

> names<-featureNames(sigGenes)
> row.names(names)=names$tracking_id
> sigGenesNames <-as.matrix(names)
> sigGenesNames <- sigGenesNames [,-1]
> sigGenesData<-diffData(sigGenes)
> row.names(sigGenesData)= sigGenesData$gene_id
Error in `row.names<-.data.frame`(`*tmp*`, value = c("XLOC_002638", "XLOC_002638", :
duplicate 'row.names' are not allowed
In addition: Warning message:
non-unique values when setting 'row.names': ‘XLOC_002638’, ‘XLOC_005270’, [... truncated]


Hence, I started to look into the ways to get Ensemble ids and the threads lead to lot of confusion and queries.

  1. should i use gtf file crated by cuffmerge or cuffcompare as input for cuffdiff ?

  2. while creating database using cummerbund should we use genome and gtf file?

  3. If the answer of 2nd question is yes, then should I use gtf from Ensemble or cuffmerge or cuffcomapre?

  4. how to get Ensemble id instead of XLOC id.

  5. Is there some error in my protocol?


Thanks in advance.
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