I've sent my plasmids for whole plasmid sequencing using the Oxford nanopore sequencing method.
All of them were assembled as monomers, but in the reads (see attached pictures), there are even longer reads, some with double or triple size of monomer plasmid, albeit at much lower rate. Should I worry about my plasmids, or how does it happen?
Also, all of my plasmids miss about 2-5 nucleotides in comparison to the expected sequence. Is that real or is that some artifact?
Thank you
What species/strain you used for the plasmid propagation?
oligomer formation can be caused by recombination, some type of e coli can be used to minimize this thing, such as DH5/TOP10 or another cloning strain.
Jonathan Jonathan Hi, they were grown in E. coli DH5alpha.
I meant more, whether based on the results, I should worry or whether is is normal.
All plasmid extractions contain some genomic DNA contamination which can show up in your reads. Plasmid multimers also happen to some extent, even in recA- strains. You're always going to get some random things showing up in the histogram. They're only a worry if they make up a substantial proportion of your obtained sequence.
As for the missing nucleotides, that could either be real or an artifact, depending on the sequence. Nanopore sequencing has a lot of trouble resolving homopolymers (ie. AAAAAAAA or GGGGGGG), as well as certain secondary structures and dinucleotide repeats. It can take very high read coverage for a confident base call in those sequences. I would check to see where the missing nucleotides are before making any conclusions.
Tomáš Hluska
Well, since DH5 is already recA- it should minimize recombination, but as Alexandra Johnson said, the multimer also happen even in recA- strain. I personally for 3 years only experience it once in TOP10 with pET-21b(+), and im using it for protein expression, there is no difference. so i guess you are ok with that.
for the question about your plasmid miss 2 to 5 nucleotide, is the nucleotide that are missing is a repeat? such as AA, CCC, TTTT, if yes please remember that ONT algorithm for basecalling sometimes can interpret TTTT signal to TT or TTT (because of the repetition)
Jonathan Jonathan thank you for your answer. No, they are single nucleotides, but they are in the same places in multiple plasmides derived one from the other, so it's some old mutation that nobody noticed so far. Also TAT triplet in bom region, but that is obviously known in the community.
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