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Hello everyone! My name is Kramer Kaplan, and I am a current graduate student working on fish genetics. My advisor and I are learning how to go through the process of 2D sequencing to obtain the entire mitochondrial genome of two species of freshwater stingrays. I wanted to submit a post here with several questions that I had. We have been using a PCR Barcoding Kit (EXP-PBC001) that has the twelve barcodes and barcode adapter mixes (BCA). I was wondering when you use that adapter mix because it does not say anything about that in the protocol. I'm following the "PCR barcoding amplicon sequencing for the MinION device using SQK-NSK007." As I have been following this protocol, the issue I have been seeing is my concentrations of DNA keep getting less and less until I have less than 20ng/ul. Before prepping the MinION, I am supposed to have around 150ng/ul of material. My first found PCR product showed about 10kB on a gel, and the concentrations were high enough, so I truly do not know what the issue is. If anyone could shed some light on these issues or offer any advice, that would be greatly appreciated!
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Hi! You can ligate the BCA to any DNA and then do a barcoded PCR, e.g if you want to use genomic DNA or have amplicons without the ONT pcr-priming tail. We also tend to get lower than recommended yield of DNA after the final streptavidin bead purification, but I think 20 ng/ul is still high. If you only get 20 ng in total then something is likely wrong, perhaps adaptors are too old or your starting concentration was off.
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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