[RickC7]

RNA-Seq and 5S

Hello: the information we have with Ribo-Zero abd Mycobacterium tuberculosis is this:

"“RZ Bacteria - (Mycobacterium tuberculosis) Efficiently removes 23S, 16S and 5S rRNAs.”

Olaf

I am working on an RNA-Seq project and on epibio website under species compatibility it states for Mycobacterium tuberculosis "will not remove 5S". Looking at Agilent results I have to agree b/c we see a very distinct peak for the 5S region after using Ribo-Zero Magnetic Kit (Gram-Positive Bacteria) Cat. No. MRZGP126. 23S and 16S are gone. My concern is that a large portion of our reads from sequencing will be 5S region and we will reduce reads for the important stuff. Has anyone been successful at removing the 5S for such an experiment? Or do you just sequence more to achieve the desired depth?

I even see my library size range shifted. Usually I expect more reads >200bp, but in this case I have an increased distribution of fragments <200bp. I believe this to be the 5S region being fragmented even smaller and being carried through the rest of Library construction protocol. SoliD RNA-Seq standard protocol. I am very hesitant to proceed with sequencing. Does anyone have a similar experience?

Thanks.

[Olaf Blue]

Generally RNA can be further enriched by size selection; I believe that 5s rRNA (about 110-120 nt) can be removed using a standard Qiagen RNEasy MiniKit as the kit will not retain any RNAs less than 200 nt. We almost never see any fragment size shift as the capture probes only interact/remove rRNA and do not affect the RNA quality in terms of insert length. What kit/method are you using for library prep?