Oxford Nanopore should have kits for direct RNA sequencing available later this year, and have several examples of how these might be used on their website. The method presented at London Calling (see OmicsOmics coverage), is primarily for mRNA, but it likely to be adapted for other RNA species in due course.
One of the ideas I've briefly thought about is using the MinION to perform ribosome profiling - a basic method would involve ligating adapters to RNA after cell lysis so Ribosomes are fixed to mRNAs with cyclohexamide treatment. Fast mode sequencing would identify the 3' end and the transcript, then sequencing speed would be massively ramped up to zip the mRNA through the pore; the bound ribosomes should cause sequencing to stall allowing counting of stall events and therefore the number of ribosomes attached to an mRNA.
Nanopore ribosome profling: In the figure above the cells from two distinct populations would be sorted (A) and mRNA with ribosomes still attached would be extracted (B). mRNA's are ligated with nanopore sequencing adapters (C). Nanopore sequencing shows distinct pause's at ribosome locations allowing estimation of quantitative translation (D). This is very much an idea that has not gone much further than the back-of-an-envelope and many issues need to be considered in developing methods like this:
- Ribosomes might not survive the ONT library prep
- Ribosomes may be too firmly attached
- RNA may "slip" through the ribosome with no effect on sequencing
- Pauses in sequencing may be sequence specific
- RNA modifications may add all sorts of challenges
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| Figure A from Smith et al 2015 |
tRNA-seq: Mark Akeson's group recently published work on MinION tRNA-seq where they tailed the tRNA with a leader-adapter such that the molecule unzipped from its usually highly configured 2D structure as it was sequenced through the pore (possibly it even reassembles on the other side of the membrane). Other RNA species are likely to benefit from methods similar to this including perhaps ribosomal profiling.
I think data like the tRNA-seq paper, and ideas like mine above and an earlier rRNA-seq idea, demonstrate how much more we may be able to do on a Nanopore sequencer than on our current short-read Illumina sequencers.
The future is very difficult to predict - but it is fun to try!
Hi Andrew, did you see Clive Bron's most recent Nanopore update where he described "blob counting"? This sounds very similar to what I'd proposed here so you might be able to do this kind of experiment much sooner than I thought.
ReplyDeleteIt may be that a "fast" mode would allow rapid ribosome footprinting for large numbers of samples, whilst a "slow" mode could be used to get sequence information as well to understand isoform translation.
Cool stuff indeed!
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