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scottdaniel 04-30-2012 09:40 AM

Read runs and technical replicates
 
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So I've downloaded some example data from SRA archives at http://www.ncbi.nlm.nih.gov/sra?term=SRA011001 and had a question about technical and biological replicates (see attached picture for a snapshot of the downloads). I know that there are two biological replicates for control and treatment conditions (in this case a miR155 over-expression retrovirus) but I'm not sure about the technical replicates.

Do read runs count as technical replicates?

And, if so, how can I use that in my analysis pipeline (currently I'm using the Tuxedo suite (i.e. tophat > cufflinks > cuffmerge, etc.)).

Right now I think I'm just telling the program that there are six biological replicates of each condition (i.e. 6 x miR155oe vs. 6 x control) which is probably wrong.

Thanks, Scott

chadn737 04-30-2012 10:38 AM

It has been shown in many cases that Biological variation is far greater than technical variation in Illumina sequencing and typically individual runs are pooled together and treated as one sample. Ideally thats why you multiplex all your biological replicates and spread the sequencing across multiple lanes.

scottdaniel 04-30-2012 11:12 AM

Quote:

Originally Posted by chadn737 (Post 72165)
It has been shown in many cases that Biological variation is far greater than technical variation in Illumina sequencing and typically individual runs are pooled together and treated as one sample. Ideally thats why you multiplex all your biological replicates and spread the sequencing across multiple lanes.

Is there paper where this is shown?

So perhaps I should merge my .sam files per biological replicate and then do the tophat/bowtie alignment?

Thanks.

chadn737 04-30-2012 11:49 AM

Marioni, J. C., C. E. Mason, S. M. Mane, M. Stephens and Y. Gilad,
2008 RNA-Seq: an assessment of technical reproducibility and
comparison with gene expression arrays. Genome Res. 18: 15091517.


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