Hi dear researchers,
Just want to ask whether you are using Minion for sequencing. I'm going to sequence a whole gene (~8kb) with it. If I want to combine a few samples and sequence together in the flowcell, how to label them? Do you add barcode to primers during the PCR phage? Or there are other ways to do this?
Hope for your reply!
Hi,
Shuai chen, I have done it by illumina sequencing platform. I do add barcode in the primers and it works well. So I think it will work well in Minion.
Good luck!
Here is something that you may be interested in. I do not have any experience with trying this or know any of the authors directly. Just could be of potential interest to you.
Real time selective sequencing using nanopore technology
The Oxford Nanopore MinION is a portable real time sequencing device which functions by sensing the change in current flow through a nanopore as DNA passes through it. These current values can be streamed in real time from individual nanopores as DNA molecules traverse them. Furthermore, the technology enables individual DNA molecules to be rejected on demand by reversing the voltage across specific channels. In theory, combining these features enables selection of individual DNA molecules for sequencing from a pool, an approach called "Read Until". Here we apply dynamic time warping to match short query current traces to references, demonstrating selection of specific regions of small genomes, individual amplicons from a group of targets, or normalisation of amplicons in a set. This is the first demonstration of direct selection of specific DNA molecules in real time whilst sequencing on any device and enables many novel uses for the MinION.
http://biorxiv.org/content/early/2016/02/03/038760
Essentially what they propose is that you can potentially "select for" the sequences you want from a pool of sequences. Thus, you may be able to accomplish what you want without even doing the PCR. Give it a read.
Dear Falseplanet,
Did you choose the barcodes sequence yourself? I mean are there any principals of choosing barcode? What should be the length or what the feature of the sequence pattern?
And where did you PCR from? For me I need to PCR this 8kb fragment from gDNA with just one pair of primers. It's very difficult to deal with the primers. Because now it's 25bp, if I add barcode to the primer, the Tm will be very high, which is higher than the recommended elongation temperature of the long PCR kit(68C). So I want to make the barcode as short as possible and at the same time make sure it will not mismatch to other sites in genome.
Dear James,
I haven't read this paper but based on what I know about the Minion, it's better to read directly. It's very hard to change the voltage and it may generate some errors. Anyway, I will read this to see what they did. Thanks!
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