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Ribo-Zero rRNA removal for RNA-seq with FFPE samples

September 20, 2012

We (which of course means someone else did most of the work and that person is Andrija Matak), made RNA-seq libraries from some FFPE tumor samples, which had been sitting in the biobank at the Medical Univeristy of Graz in Austria for 6 years.  We used the Ribo-Zero magnetic rRNA removal kit to deplete the samples of rRNA.  The whole experiment worked surprisingly well.  All the samples had less then 10% rRNA reads and most were down around 2 %.  A couple of the samples had very poor alignment (less then 20% of reads) and had to be discarded from the analysis.  The percentage of reads aligned in general was significantly lower (around 45 to 60%) then what I’ve been seeing with high quality RNA (usually above 70%).  It also seems like less of the aligned reads were being assigned to genes by HTseq-count.  But with all that being said the experiment went very well.  The data matches the data from a bunch of genes we (again not meaning me) have checked by in situ hybridization.  The variability within each tumor region was remarkably low.

Take a look at the DESeq output. Pretty clean.  I was a little surprised.

BTW, that is the QC report that ezDESeq.sh makes for you.  All you have to do is make two folders with your fastq files and run the script.  It’s that easy.

Finally, here was my adaptation of the Ribo-Zero protocol and how I pipe it into the TruSeq RNA protocol.  If I were to do it again, I would divide all the Ribo-Zero volumes by four.  Here I started with 1 μg of RNA and only had to do 9 cycles of PCR amplification, so clearly there is some room for savings here.  One other thing, the official Ribo-Zero protocol says you should ethanol precipitate, which is way too old-school.  They also say you can use AMPure RNA beads.  I guess they don’t realize this but AMPure beads precipitate all nucleic acids, so you can just use regular AMPure XP beads (or better yet homemade) so there is no reason to buy another expensive reagent to precipitate your RNA.

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17 Comments
  1. Ming Dong permalink

    nice blog, useful stuff. Now I am learning ChIP-seq, and probably will do it next week. I wish I could find this site earlier.

  2. ethanomics permalink

    Hi Ming,
    Thanks for the compliment. Let me know if you have any questions or troubles as you work through your experiment.

    Ethan

  3. Ming Dong permalink

    Can you construct good library from non-PAGE/desalted purified oligos as adaptor? Do you purify your oligos now?

    I ordered some illumina single strand oligos from some third party vendor, it’s only “desalted”, not “PAGE purified”, and I annealed them together. The adaptors can ligate to A-tailed mock DNA fragments (I ligated them to 250 bp DNA fragment, got 350 bp product (250+two adaptors), and the 250 bp band disappear. ) Before go to real world DNA sample, I was wondering whether I should use PAGE to purify the oligos. What’s your opinion?

    Thanks

  4. ethanomics permalink

    I always just get them “desalted” from IDT. I hard to imagine a way in which a very small percentage of n-1 oligos could possible have an adverse effect on sequencing. Some people get them HPLC purified but it’s just paranoia I believe.

    I would suggest that you do NOT resuspend them in water. DNA should be stored in a pH buffered solution. I use 10 mM Tris pH 8.0, 0.1 mM EDTA. Resuspending your oligos in water is well documented to significantly reduce their stability.

  5. Ming Dong permalink

    Thanks, basically, your “desalted” oligos also work fine for library construction?

    —“I hard to imagine a way in which a very small percentage of n-1 oligos could possible have an adverse effect on sequencing.”

    I don’t know the percentage, esp. for a ~60 nt oligo.

  6. Ming Dong permalink

    HI, I almost decided to use desalted homemade oligo as adapter until this paragraph from Sigma freaks me out:

    http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/best-purification.html

    PAGE
    The basis of the PAGE separation… resulting in purity levels of 95–99% full-length product. …PAGE is the recommended purification for longer oligos (≥50 bases).

    I am using Illumina Adaptors, so ~63 bp. I am sure you got good results with desalted (non-PAGE purified) oligos, right?

    How long are your oligos?

    thanks

    Ming

  7. ethanomics permalink

    Hi Ming, I get my oligos from IDT and they do mass spec QC on all of them. When I order the adapters using standard desalting they are at least 99% full length product (they recommend HPLC purification but I think it is to make money). Furthermore a small percentage of n-1 product is not going have an adverse effect on your library. It’s not like making an expression plasmid where you will get a frameshift mutation which will mess everything up. Most likely most n-1 oligos will not cause a problem, then few that do will either not amplify (not a problem), not stick to the flow cell (not a problem) or not sequence (not a problem). It’s an expensive experiment so it just seems good to do it the most expensive way, but it really is not necessary and my guess makes zero difference in the data generated and even if it does make a difference it will be so small compared to other factors.

    Worst case scenario, which I would guess is highly unlikely, you lose say 50,000 reads because the adapters have some n-1 product. The money you save will allow you to sequence several more samples. The first being an unlikely and negligible effect and the second being a nearly certain huge improvement in your data set.

    The oligos I’m using are the TruSeq adapters as written at the end of the protocol.

    Ethan

    • Ming Dong permalink

      Thanks, I feel better.

      I’am using a local oligo vender, Elimbio, while I assume all the company are using similar tech to make oligos, so the quality/percentage of full length should be similar.

      While tech support of Elimbio says 60 mer will have 50% impurity. I run an agarose gel, and see all the oligos have two bands, I don’t know it’s the truncated form of oligo or different DNA conformation though (a denaturing PAGE will be more revealing)

      Have you run a gel of your oligos?

  8. ethanomics permalink

    You would need to run them on a denaturing (urea) acrylamide gel (old school ‘sequencing gel’). We use to do this a long long time ago. The two bands could be secondary structure differences. That being said, if their 60-mers are only 50% pure I would switch to IDT. Here is the mass spec analysis of a 60-mer I just ordered from IDT at 25 nmole scale with standard desalting.
    https://ethanomics.files.wordpress.com/2012/10/108646721_0_1.pdf
    As you can see it is >99% pure.

  9. Ming Dong permalink

    Thanks, yeah, 50% and 99% are different story.

  10. Ming Dong permalink

    I am reading your homemade Ampure beads protocol, it seems that you have perfectly decoded /reversed engineered Ampure beads. Great Job!

    If I understand correctly, Ampure beads itself is nothing mysterious, the key is in the buffer, i.e PEG 8000 + NaCl, right?
    It’s PEG that precipitates DNA onto the beads, and using different beads to DNA ratio to select actually is using different PEG concentration to selectively precipitate DNA of different size.

    So we can reuse Ampure beads by add PEG+NaCl buffer right?

    I need to bind crosslinked DNA (~200 bp, with protein on it) to somewhere, then do several enzyme processes. Between processes, I got do buffer exchange, i.e. clean out previous buffer then add new buffer. I think I can use AMpure + PEG-NaCl buffer to do it.

    What’s your opinion?

    BTW, I am in Berkeley, near SF, if you visit bay area, I can show you around. You really helped me a lot.

    Thanks

    Ming

    • ethanomics permalink

      Hi Ming,
      It wasn’t me that made that protocol. Nor did I even transcribe it. So I’ll say thanks to the guys that did.

      I have never re-used the beads, but I don’t see a reason why you couldn’t if you are doing a sequence of enzymatic steps. But that still doesn’t mean it would work, but it does sound like a good cost saving option. And perhaps easier then adding new beads. I think it is a good idea, but again that still doesn’t mean it would work.

      I am a SF native and I still miss it (except the fog and cold ocean). I’m still sort of hoping to land back there some day. I’m just taking the long way back from Boston via Greece and Australia.

      Ethan

      PS sorry for the delay in responding!!

  11. Ming Dong permalink

    using different beads to DNA ratio to select actually is using different PEG concentration to selectively precipitate DNA of different size.

    I mean “different beads to DNA ratio to select actually” actually has nothing to do with beads, it’s the different BUFFER volume that works, it’s the PEG in different volume of buffer, which makes different PEG concentration in DNA beads mixture, and it’s different PEG concentration that selectively precipitate DNA by different size.

    • ethanomics permalink

      Correct, it is all about the PEG concentration (and I guess to some extent the NaCl concentration). I routinely use 1 volume AMPure and 1 volume 30% PEG800/1.25 M NaCl to precipitate small DNA fragments (>100 bp).

      See this SEQanswers thread
      http://seqanswers.com/forums/showthread.php?t=3752

      One thing I’m just figuring out is that one problem with AMPure is that it just washes away the unwanted material whereas QIAquick and phenol-extraction denature the unwanted proteins prior to removal. So you can imagine in some cases AMPure might not work when trace amounts of enzyme could cause problems.

      In a specific case I was working with, I did PCR, AMPure purified, followed by a 4 hour restriction digest at 55˚C. The subsequent ligations failed most like because trace amounts of the polymerase was chewing and probably blunting the ends of my DNA fragments. This problem was eliminated when I purified the PCR reactions with QIAquick columns. I’ll post a little more about this with some examples sometime in the near future. But it’s something to keep in mind.

  12. Ming Dong permalink

    Hi, Ethan,

    Thanks for your reply, I didn’t know you are a SF native. If you come back to SF, I will buy you lunch.

    I first update my results:
    1. I can reuse the beads. I did with beads that reuse only once, and the performance is as good as new beads.
    2. IN the following paper, the author reused the same bead again and again for stepped enzyme reactions.

    “The SPRI beads are added to the sample after the shearing step, and remain in the reaction vessel throughout the sample preparation protocol. By allowing each cleanup step to employ the same beads, the with-bead method greatly reduces the number of liquid transfer steps required. ”

    Fisher et al. Genome Biology 2011, 12:R1, A scalable, fully automated process for
    construction of sequence-ready human exome
    targeted capture libraries

  13. Ming Dong permalink

    Ethan,

    1. In your homemade beads protocol, you have two PEG buffer,
    a. 20% PEG+2.5 M NaCl
    b. 1 ml beads plus 18% PEG, 1 M NaCl, 10 mM Tris, 0.05% Tween 20, 1 mM EDTA
    why the two buffer are different?

    2. Do you have some idea about what’s the lowest DNA concentration Ampure can catch/precipitate? How does it compare to EtOH precipitation?

    THanks

    Ming

  14. Neeraj Sethi permalink

    Hi there,

    Can I also ask if you have any experience of Ribozero Gold? Is this worth the extra money as Ribozero is expensive enough already!

    thanks

    Neeraj

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