Calling on #ONT #Nanopore #sequencing experts: when sequencing through a stretch of 120nt dA using a DNA plasmid kit (outsourced in our case), what should one expect as average length in the output? The loss we are seeing is a bit extreme #RNAsky
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Thanks Mark, this is really helpful! In the original consensus sequence that we received it was 35nt. After reanalysis and raw data being provided, we are now at an average of 79nt. Your comment is giving me more confidence that it is actually all fine. We are working on other QC methods in parallel
I would also like to know if you prepared your library from linearised or supercoiled plasmids? Did you use a rapid or ligation sequencing kit? And did your poly(A) tail incorporate a (non-A) linker sequence? Sorry for the 20 questions - they all count!
We observed ~20% deletion error rates in the poly(A) tails of our plasmids when we sequenced using RBK114 (Rapid) kits. This is pretty approximate though - direct RNA or cDNA sequencing, then poly(A) tail length analysis with Dorado is more reliable (it corrects for homopolymer errors) but $$.
We detected sub-clonal poly(A) truncations in mRNAs transcribed from plasmid templates using this method, and have had more consistent results with PCR-based templates, as per @sethcheetham.bsky.social's reply. Dorado poly(A) analysis needs to specify any non-A linker sequences to be accurate.
Thank you Helen, this is really helpful to know! If using a cDNA seq protocol significantly improves the results, we may want to go down this route even if $$. I will have a look into that; shouldn't be difficult to adapt for plasmids. Thanks again!
Thanks! We have outsourced this, so I would need to enquire about technical details. We provided supercoiled plasmid. The tail did contain one non-A base if that is what you are asking? This one was lost too.
I have heard about this issue elsewhere as well. Loss of poly with plasmid sounds very interesting to me given how repetitive sequences and plasma is really don’t like to mix in general. It is something synthetic biologist need to really learn and improve upon.
Thanks both and yes, absolutely agree. We use NEB Stable E.coli to reduce the loss of repetitive sequences, but we seem to have lost ~90nt. PCR is not an option for us at this point due to amount of product we would need. We are looking at RCA in the future.
We have used NEBstable a lot but not seen any difference to other cells (NEB 10beta) in terms of stability. Sadly we have not characterised it in depth, and the issue is for repeated units rather than long A stretches. But we found growth at 30C rather than 37C helped (NEBstable did not help!)
How much mRNA are you producing? We routinely use PCR to generate templates for up to 1-20mg productions. I know people that have done larger batches too.
For routine production, we are usually more in the 30-50mg range. I should say we do IVT in flow, not batch. How are you dealing with PCR errors at this scale?
There are some tweaks like segmented polyA tails that somewhat reduce deletions, but they're still very frequent. Worse, they are sometimes subclonal so sanger sequencing doesn't even detect the deletion. The field definitely needs better synthetic biology solutions. https://doi.org/10.1261/rna.069286.118
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For a 120nt polyA encoded in a plasmid, we would expect something around 40-50 in the output.
https://doi.org/10.1261/rna.069286.118