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I was just wondering if anyone has used the minIon for plasmid sequencing? I suppose it might be overkill and a waste or reagents for such a small task. But I am asking because I have large plasmids to sequence (in the 18 to 20 kb range), and the plasmids contain several copies (up to as many as 4 copies) of certain sequences. So using sanger sequencing is costly and labor intensive and we end up with reads that could align to more than one spot on the plasmid making it hard to tell if and where a mutation has occurred. I am also exploring Illumina based methods, but am curious about the nanopore technologies. Seems like a 20 kb plasmid could simply be linearized and the entire molecule sequenced in a single read.
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Have a look at methods for Zika virus sequencing project.
This sounds very similar to zika sequencing project, and should work reasonably well (provided it is medium GC% sequence).
Ideally one would like to make partial digest of the plasmid, aiming to cut most of them once or twice, and make 2D library from it.
PS: keep in mind the ~0.1 - 1% systematic error rate remaining after assembly consensus steps. It would really depend on the actual template sequence.
Hopefully there are no high GC hairpins in the sequence, because they would stop the complementary read (which goes from single stranded template).
So you may have some bits that have very low quality hear the hairpins. -
Hey, did you ever get this working? I am thinking about creating a single cut in the plasmid and sequencing entire AMR plasmid. Know any papers where this has been done?Comment
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Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
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Introduction
Single-cell sequencing technologies have undergone remarkable advances over the past decade, transitioning from low-throughput experimental approaches to highly scalable platforms capable of...05-22-2026, 06:42 AM
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