ribozero kit problem

[maggielaw]

Hi All,

Anyone is facing instability of rRNA removal using ribozero kit of Illumina? Thanks.

[dpryan]

If by "instability" you mean that the you often get a relatively large percentage of rRNA sequenced after using the kit, then yes, we see that all the time. We've tried a bunch of different kits and the results are hit or miss. I think we only get good results if the kits are used more or less immediately after RNA extraction.

[pmiguel]

It usually works okay for us. The biggest consistent failure mode is that if you don't use a sample with enough rRNA, the rRNA capture probe oligos can bleed over into your library. You can tell if they are the source of your "rRNA" contamination because they will map to the wrong strand of your rRNA reference.

So we insist people bring us at least 1ug of total RNA when using this kit.

--
Phillip

[dpryan]

@pmiguel: I hadn't thought about checking the strand, that's a good idea! What sort of left-over rRNA levels do you tend to get? Ours range from low (<0.5% rRNA) to absurdly high (up to 20% or so).

[pmiguel]

@pmiguel: I hadn't thought about checking the strand, that's a good idea! What sort of left-over rRNA levels do you tend to get? Ours range from low (<0.5% rRNA) to absurdly high (up to 20% or so).

As long as they are below 30% or so I call it a win. (Some plant tissues produce total RNA that seems to be >95%rRNA). But I think we probably are getting similar results to you most of the time.

The samples that showed the wrong strand hits produced >90% rRNA reads.

Oh, the other "trick" -- only buy the Ribozero "Gold" library kit. This is for "human, mouse, rat" but we've used it for other animals, including moths. If you get the non-Gold kit, there are no probes to the mitochondrial rRNA. Fine for some tissue--but disastrous for others. (Eg, muscle tissue.)

Fortunately the plant Ribozero library construction kit includes probes to nuclear, mitochondrial and chloroplast rRNA.

--
Phillip

[dpryan]

Our sequencing folks were getting worried whenever the numbers were greater than the 2% advertised levels. Glad it's not just us (not that I'm surprised, the folks in our sequencing facility are really good).

[maggielaw]

It usually works okay for us. The biggest consistent failure mode is that if you don't use a sample with enough rRNA, the rRNA capture probe oligos can bleed over into your library. You can tell if they are the source of your "rRNA" contamination because they will map to the wrong strand of your rRNA reference.

So we insist people bring us at least 1ug of total RNA when using this kit.

--
Phillip

Hi Philip,

Which kits are you referring to? TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat (Cat# RS-122-2201)? TruSeq Stranded Total RNA with Ribo-Zero Globin(RS-122-2501)? or others?

[pmiguel]

Hi Philip,

Which kits are you referring to? TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat (Cat# RS-122-2201)? TruSeq Stranded Total RNA with Ribo-Zero Globin(RS-122-2501)? or others?

I guess this would be an issue for all these kits. But the one we use most frequently is:

TruSeq Stranded Total RNA with Ribo-Zero Gold, Set A Indexes (Cat# RS-122-2301)

and/or

TruSeq Stranded Total RNA with Ribo-Zero Gold, Set B Indexes (Cat# RS-122-2302)

--
Phillip

[subxero]

It usually works okay for us. The biggest consistent failure mode is that if you don't use a sample with enough rRNA, the rRNA capture probe oligos can bleed over into your library. You can tell if they are the source of your "rRNA" contamination because they will map to the wrong strand of your rRNA reference.

So we insist people bring us at least 1ug of total RNA when using this kit.

--
Phillip

Is this the only solution you tried, increasing the input?

We are having this problem right now but we have very limited amounts of RNA so are hands are a bit tied as far as the amount of input RNA we can use.

Is it possible to decrease the amount of probes used or something along those lines?

[pmiguel]

Actually, I re-thought this recently. The ribo-zero probes (biotinylated oligonucleotides) would not be removed more effectively if they are double stranded -- the streptavidin beads are what removes them--duplex or not.

Also, the cDNA produced by 1st strand synthesis, would be the same strand as the ribozero probes. So telling the two apart would require the ability to distinguish human from mouse and rat (or whatever species your RNA doesn't come from) to be sure it is ribozero probes that are contaminating your sample.

I was thinking this morning that what we needed was a few sets of rRNA primers so one could assay RNAseq libraries for rRNA levels directly with qPCR prior to sequencing.

--
Phillip

[chenyudr]

Actually, I re-thought this recently. The ribo-zero probes (biotinylated oligonucleotides) would not be removed more effectively if they are double stranded -- the streptavidin beads are what removes them--duplex or not.

Also, the cDNA produced by 1st strand synthesis, would be the same strand as the ribozero probes. So telling the two apart would require the ability to distinguish human from mouse and rat (or whatever species your RNA doesn't come from) to be sure it is ribozero probes that are contaminating your sample.

I was thinking this morning that what we needed was a few sets of rRNA primers so one could assay RNAseq libraries for rRNA levels directly with qPCR prior to sequencing.

--
Phillip

I think the mentioned "a few sets of rRNA primers" are necessary tools for checking rRNA removal efficiency.

[dwong]

Have you guys ever considered using NuGEN's InDA-C customized depletion? They also have standard depletion kits for human, Drosophila and mouse. The depletion is only performed after the library is actually made. Just a thought.

[pmiguel]

Have you guys ever considered using NuGEN's InDA-C customized depletion? They also have standard depletion kits for human, Drosophila and mouse. The depletion is only performed after the library is actually made. Just a thought.

Yes, I've thought about it. We've actually had a customer that used it to deplete a complex environmental (insect gut flora) RNA sample set of prokaryotic and eukaryotic rRNA.

--
Phillip

[dwong]

I used their Drosophila kit and it knocked down the rRNA quite a bit. Its great that its specific to the organism of choice, but that's just my personal opinion

Darren

[kmcarr]

Have you guys ever considered using NuGEN's InDA-C customized depletion? They also have standard depletion kits for human, Drosophila and mouse. The depletion is only performed after the library is actually made. Just a thought.

NuGEN will also work with you to create a custom set of oligos to use with this kit. These can be either for rRNA from a non-model organism or any other RNA you would like to deplete from your library (e.g. globin transcripts in blood). Of course you need to know the sequence of the transcripts you wish to design oligos for.

A client of ours had NuGEN add some custom oligos for some specific targets they wanted depleted in a host/pathogen mix which reportedly worked well.