Question: How To Combine Technical And Biological Replicates In Rna-Seq Studies?
4
gravatar for camelbbs
6.8 years ago by
camelbbs660
China
camelbbs660 wrote:

Hi,

I want to ask in analysis of rnaseq data to detect differential expressed genes, etc., can technical replicates and biological replicates put together to compare?

Thanks,

Ch

rna-seq • 4.7k views
ADD COMMENTlink modified 6.8 years ago by Istvan Albert ♦♦ 81k • written 6.8 years ago by camelbbs660
3
gravatar for Steve Lianoglou
6.8 years ago by
Steve Lianoglou5.0k
US
Steve Lianoglou5.0k wrote:

The standard recommendation is to combine your technical replicates into one "lane" by simply adding their reads together. Use your biological replicates to estimate variance/dispersion.

ADD COMMENTlink written 6.8 years ago by Steve Lianoglou5.0k

Since I don't have biological replicates for one sample, so I want to use technical rep for analysis, I don't know if that is also working

ADD REPLYlink written 6.8 years ago by camelbbs660
1

If you have, say two conditions, with 2 biological replicates in condA but no biological replicates in condB, there are ways to accommodate for this. Read through the edgeR and DESeq vignettes to get an idea. The answer is not to use your second technical replicate as a biological one.

ADD REPLYlink written 6.8 years ago by Steve Lianoglou5.0k

But I am not sure if the pvalue from partial replicate samples are believable

ADD REPLYlink written 6.8 years ago by camelbbs660

Then sequence another biological replicate?

I'm not sure where you want this conversation to go ... I'm just pointing you to where I think you'll find a reasonable thing to do with your data explained within a tutorial.

ADD REPLYlink written 6.8 years ago by Steve Lianoglou5.0k

Thanks a lot! I think you gave very reasonable suggestions.

ADD REPLYlink written 6.8 years ago by camelbbs660

A little bit confused here. "simply adding their reads together" means add the read counts after tophat and htseq or merge the raw data (fastq) into a single file, then go through tophat and htseq ?

ADD REPLYlink written 6.4 years ago by chunxuan10

means add the read counts

ADD REPLYlink written 6.4 years ago by camelbbs660

If you already aligned both datasets separately, merging the two bams together then doing feature counting via ht-seq on the merged bam would get you the exact thing I'm suggesting to do if you have technical replicates. There are other ways to get equivalent counts, and if those are easier for you to do, then do that but hopefully you get the basic idea.

ADD REPLYlink written 6.4 years ago by Steve Lianoglou5.0k
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