minIon for plasmid sequencing

CHObot · 09-23-2016, 06:28 AM

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.

Markiyan · 09-26-2016, 03:07 AM

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.

nanomonster · 10-05-2017, 06:25 AM

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?

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09-23-2016, 06:28 AM	#1
CHObot Junior Member Location: Greater Boston Area Join Date: May 2013 Posts: 8	minIon for plasmid sequencing 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.

09-26-2016, 03:07 AM	#2
Markiyan Senior Member Location: Cambridge Join Date: Sep 2010 Posts: 116	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.

10-05-2017, 06:25 AM	#3
nanomonster Junior Member Location: Scotland Join Date: Oct 2017 Posts: 1	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?