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(first question on the forum 
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I am looking for some ideas and brainstorming on RNA-seq sample prep and data analysis.
I am working with low-input RNA samples (1 ng/uL if I am lucky) and from that RNA only a very tiny fraction is of my interest (viral RNA). I’ve been testing an approach in which I first rRNA-deplete my sample; then amplify the total RNA and then perform sequencing. The goal is to remove RNA of no interest in order to increase the representation of RNA of interest. The idea is that after rRNA depletion, a higher fraction of RNA of interest would be present in the sample and amplified by the NuGEN kit. Straightforward.
I had the following experimental set-up: DNase-treated RNA sample -> rRNA depletion (2 kits tried-out: Ribo-Zero and NEB next for human rRNA) -> ovation NuGEN kit -> QC on Bioanalyzer -> nextera library prep -> illumina sequencing (Miseq)
There was also a control sample (in duplicate) on which rRNA depletion was not performed.
Results: The sequencing data indicated that while there is much less rRNA compared to the control samples, the fraction of RNA of interest hasn’t improved (= pretty much same ‘% of reads of interest’ between rRNA depleted samples and control samples).
The DNA output from the NuGEN kit was good, suggesting that even though the depletion was performed on low input RNA; there was enough input to NuGEN to grant good amplification.
When I map the obtained reads to rRNA (human & mitochondrial) I indeed see that there are less reads that correspond to this rRNA compared to control samples. What seems to be happening is that some other ‘RNA of no interest’ got amplified instead (e.g. bacterial rRNA or still undetermined other RNAs) but my target of interest did not get any boost in amplification
It seems to me that the experiment went OK but I am looking for ideas on why the results are like this and maybe for improved experimental set-up.
Here are some of my thoughts:
- The extra low input of RNA to NuGEN make the amplification procedure less efficient resulting in a similar amount of target of interest with or without rRNA depletion
- The expected ‘improved’ concentration of target of interest is not significant enough to see the results back in sequencing data (e.g. say the initial fraction of target in the sample is 0.005%; after depletion 0.05%; just speculating)
- Biased amplification ??
Do you guys think my thinking is correct or what could be else happening? There is no contamination or degradation going on.
I am wondering if there are some people here who also work with low-input RNA samples and tried rRNA depletion. Or maybe there are people who tried some enrichment/depletion approaches that are executed after library prep ?
)I am looking for some ideas and brainstorming on RNA-seq sample prep and data analysis.
I am working with low-input RNA samples (1 ng/uL if I am lucky) and from that RNA only a very tiny fraction is of my interest (viral RNA). I’ve been testing an approach in which I first rRNA-deplete my sample; then amplify the total RNA and then perform sequencing. The goal is to remove RNA of no interest in order to increase the representation of RNA of interest. The idea is that after rRNA depletion, a higher fraction of RNA of interest would be present in the sample and amplified by the NuGEN kit. Straightforward.
I had the following experimental set-up: DNase-treated RNA sample -> rRNA depletion (2 kits tried-out: Ribo-Zero and NEB next for human rRNA) -> ovation NuGEN kit -> QC on Bioanalyzer -> nextera library prep -> illumina sequencing (Miseq)
There was also a control sample (in duplicate) on which rRNA depletion was not performed.
Results: The sequencing data indicated that while there is much less rRNA compared to the control samples, the fraction of RNA of interest hasn’t improved (= pretty much same ‘% of reads of interest’ between rRNA depleted samples and control samples).
The DNA output from the NuGEN kit was good, suggesting that even though the depletion was performed on low input RNA; there was enough input to NuGEN to grant good amplification.
When I map the obtained reads to rRNA (human & mitochondrial) I indeed see that there are less reads that correspond to this rRNA compared to control samples. What seems to be happening is that some other ‘RNA of no interest’ got amplified instead (e.g. bacterial rRNA or still undetermined other RNAs) but my target of interest did not get any boost in amplification
It seems to me that the experiment went OK but I am looking for ideas on why the results are like this and maybe for improved experimental set-up.
Here are some of my thoughts:
- The extra low input of RNA to NuGEN make the amplification procedure less efficient resulting in a similar amount of target of interest with or without rRNA depletion
- The expected ‘improved’ concentration of target of interest is not significant enough to see the results back in sequencing data (e.g. say the initial fraction of target in the sample is 0.005%; after depletion 0.05%; just speculating)
- Biased amplification ??
Do you guys think my thinking is correct or what could be else happening? There is no contamination or degradation going on.
I am wondering if there are some people here who also work with low-input RNA samples and tried rRNA depletion. Or maybe there are people who tried some enrichment/depletion approaches that are executed after library prep ?
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