I recently watched a presentation on RNA-seq that covered some of the choices one can make along the way, and I didn't fully understand one of the choices in particular. Near the beginning of the process, you can choose a fragmentation method (e.g. RNA: nebulization, hydrolysis; cDNA: sonication, Dnase I treatment). Each fragmentation method is biased in its own way, and the goals of the study may influence the choice of method.

I didn't fully understand the reasons for the characteristic biases (how do they happen?), and when to choose one method versus the other (which methods work best for which goals?). Put another way, what are the mechanistic explanations for the characteristic biases that result from each method, and what is the significance as far as downstream processing and associated challenges?


The heart of this question is about fully understanding the particular choice of when and how to fragment. The reason I have not accepted an answer, yet, is that the answers provided so far address the mechanism part of the question, but not the ultimate reasons for choosing one versus the other.

  • $\begingroup$ The biases depend on fragmentation methods. DNA is usually fragmented physical shearing. (nebulization/sonication). The overrepresentation is because of the ends being more stable (easy to break a stick from the middle). There are other kinds of fragmentation methods (thermal-catalytic) and there the biases are different (modern kits claim that they have very less bias) $\endgroup$ – WYSIWYG Sep 15 '14 at 4:24
  • $\begingroup$ Bias can happen at other levels too- reverse transcription, priming, amplification etc $\endgroup$ – WYSIWYG Sep 15 '14 at 4:25

Good question, a lot of this is still being figured out. Here's what's known so far:

Fragmentation methods based on restriction enzymes aren't random.

Reverse-transcription performed with poly dT-oligomers, which bind to the 3' poly-A tails, is strongly biased towards 3’ end of transcripts.

Reverse-transcription with random hexamers results in an under-representation of 3’ ends. This is from the reduced number of priming positions at which the reverse transcriptase enzyme can start cDNA synthesis.


messenger RNA has a poli A tail at its 5´ end. thus when the poly-dT hybridize the mRNA in the reverse transcription the cDNA will carry this poly-dT at its 3`end. Thus, as reverse transcription will always start at the 3´ and of the mRNA its is more likely that this region is better rev. transcribed. Bigger the mRNA is bigger will be this concern.

About the bias related to RNA fragmentation I don't know, I can make the guess that both ends are more lost due to the fragmentation itself

watch this video from life technologies http://www.youtube.com/watch?v=0MJIbrS4fbQ


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