Detecting effects of DNA base modifications by NGS
Hi
For small bacterial plasmids, is modest Nanopore sequencing with access to raw signal files generally sufficient to assess whether a DNA base modification affects sequencing (e.g. read truncation or signal differences), or is very high-throughput PromethION data necessary even for an initial feasibility test?
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It may be best to explain what you exactly want to do and how you are planning to do it. Is there a reference available that you would use?
This is a small pilot using matched plasmid samples with and without a known DNA base modification and an available reference sequence. The aim is only to assess feasibility: whether the modification affects sequencing behaviour (e.g. read length/truncation) and whether any consistent difference is visible in the raw nanopore signal. We are not attempting full modification calling at this stage, just determining whether there is any signal footprint that would justify deeper sequencing later.
I have not heard of this being a "thing" but it sounds reasonable to try out, especially if you have a control (pos/neg) available. Since MinION's are far cheaper it would make sense to start there.
If you have access to ONT forums then ask the tech support there.
Sorry, how can I evaluate whether a DNA modification (e.g., methylation or a bulky chemical addition like a sugar) interferes with Oxford Nanopore sequencing itself? For example, should one look at read length distributions, sequencing yield, pore blockage rates, basecalling quality scores, alignment statistics, or raw signal characteristics (FAST5/POD5)? Are there accepted indicators showing that modified and unmodified samples behave similarly enough to conclude that sequencing is not being disrupted?
Thanks for any thoughts
While you will have known controls (modified and not) will you have fragments of known length? If not you may only be left with identifying if the particular base that is modified is being seen in the data (or not), which would tell you that the pore is able to go past the modification and if there is sequence beyond the modification.
You should reach out to ONT support to get their take on this. I am reasonably certain that this is something they will have looked at internally and may have specific suggestions to check on.
Thank you, yes, the whole length of an intact career is know already