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Old 04-05-2019, 03:50 AM   #1
JasperGeh
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Location: Hanover, Germany

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Default ssDNA sequencing on MinION without PCR or doublestranding

Hey people!

A colleague of mine wants to sequence a library of ssDNA Adeno-associated viruses on the MinION to get a distribution of their genome sizes and orientations.
To get the size distributions without fragmenting, we wanted to use the ligation kit from ONT which requires dsDNA. He wants to avoid a PCR step though, as to not lose the information about the genome orientations, as it would be essentially random which strand/orientation gets sequenced, right?

Is there a way to directly sequence ssDNA on the MinION? I found nothing so far, so possibly not.
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Old 04-07-2019, 03:26 PM   #2
gringer
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Is it possible to do a single cycle of PCR, i.e. the DNA equivalent of a reverse-transcription step?

I'm not sure whether you'd be able to get away with using random octamer primers - it would depend on how the sequencing adapters are ligated - but if there's a known viral priming sequence that can be used, it should be fine.
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Old 09-05-2019, 04:36 AM   #3
abbecker
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I am having the same problem. I want to feed isothermal amplification products to the MinION, but these are all single stranded and >50kb. And no, I can not use a reverse primer, because the repetitive 50kbp structure needs to stay full length and intact. Any reverse primer I apply after the isoamp would bind anywhere in the repetitive structure.
Any solutions?

Last edited by abbecker; 09-05-2019 at 04:40 AM.
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