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Old 03-10-2017, 06:00 PM   #1
Junior Member
Location: Bay Area, CA

Join Date: Apr 2016
Posts: 6
Default RNA-Seq libraries ok for DGE analysis?

Hello all,

I have spent the last several months constructing 48 libraries for RNA sequencing from developing stingray tissue, which I am hoping to sequence on a HiSeq 4000 with 100 bp PE reads.

I have been as consistent as possible throughout my library preps, but I still ended up with a range of fragment sizes after size selection (median size range from 294-485). Further, each library has a fairly broad size distribution. I've attached BioAnalyzer traces for ~ 40 of the samples.

Will these libraries be comparable enough to detect differential gene expression among them? Alternatively, are there bioinformatics tools available that can account for batch effects during processing? Fragmentation is supposed to be random, and the fragment size differences occur across treatments i.e. each treatment has replicates with both shorter and longer fragments.

Thank-you in advance for any feedback!

- John
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File Type: pdf Final_BioAnalyzer_cDNA_Libraries_ALL.pdf (4.43 MB, 29 views)
JDSwenson is offline   Reply With Quote
Old 03-13-2017, 12:35 PM   #2
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Location: California

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I don't see anything in the traces that would make me overly alarmed. Most DGE software packages (edgeR, DESeq2, etc.) allow you to specify batch variables.
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Old 03-13-2017, 03:11 PM   #3
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Location: US

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Provided one is starting with all high quality RNA-samples, RNA-seq libraries can (and should) look indistinguishable on Bioanalyzer traces. With most kits the libraries will look similar to your sample 7A. Especially if poly-A enrichment would have been used (varying degrees of 3' biases?), we would not suggest to use these libraries -- clearly varying conditions were at work during library prep. I have no DGE data from technical replicates to back up this recommendation, though.
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Old 03-17-2017, 01:51 AM   #4
Location: Montpellier (France)

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I advise you to build all the libraries you're going to compare at the same time to reduce technical biases.
Are you using a kit or a home made protocole?
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