Nanopore sequencing 101 Q&As
Here you can find all of the questions and answers asked during the Nanopore sequencing 101 webinar.
1. How many times can you reuse a flow cell?
There is a flow cell wash protocol that you may use between running one sample and the next sample(s). Depending on how rigorously you are washing the flow cell, we have seen customers using a flow cell up to five times; five is probably a good starting point in terms of what you can expect for the number of times you can reuse a flow cell. You can also run a platform QC prior to a run to evaluate how many of the nanopores are available to you before you start a run. So when you wish to reuse a flow cell, you can evaluate whether there are enough active nanopores available before you begin your sequencing experiment. Our wash kit is available from the Store.
2. What is the maximum concentration of poor quality of DNA that a nanopore sequencing platform can sequence with good sensitivity?
We get these types of question often because customers perceive that a nanopore is very small and so they might have issues with contaminants clogging the nanopores. In reality, it is hard to block a nanopore. We guide our customers through standard DNA and RNA quality control techniques, that you would typically use in molecular biology, as these are applicable to our platform. So, measuring A260/280 and A260/A230 ratios, and using the Bioanalyzer for QC, help to ensure that you have high quality samples. These are the main protocols that we recommend to our customers in order to be able to evaluate, " is this a good sample to take on to sequencing or not?"
3. Are there any special instructions on how to store a flow cell, after washing it? And how long can we store a reused flow cell?
Protocols and reagents are provided on washing and storing flow cells - please see the Store for the Flow Cell Wash Kit, and the Protocol library in the Community, for more information.
4. How many bacterial genomes can I sequence on a MinION?
The number of samples that you can run on a flow cell (let's talk about the MinION Flow Cell) really depends on your experimental design. To be able to answer this question, it will depend on the size of the genome(s), and the level of coverage you want for those genomes. Let's just for simplicity say we're looking at a single MinION Flow Cell, and we are sequencing and generating about 10 Gb of data. Depending on the size of the genome you are looking at and what level of coverage you would like, you then divide that into the 10 Gb to be able to determine what the number of samples is that you are able to process on a single flow cell. You might be in line to get more than 10 Gb data from your flow cell, which means you could choose to stop, wash the flow cell, and reuse it another time to generate additional data. We suggest looking at the Metagenomics Getting Started guide on the Resource Centre for an example calculation.
5. What is the typical yield of a flow cell?
It is highly dependent on the quality of the input DNA. A MinION Flow Cell should generate over 25 Gb of data, if good quality DNA is sequenced. Please see the Product comparison page for more information about flow cell outputs (https://nanoporetech.com/products/comparison)
6. Do you have any recommendations for DNA extraction protocols?
A fantastic question because there is obviously no one single sample type out there! There is an entire section in the Knowledge section of the Nanopore Community dedicated to extraction protocols for different biological samples. I will just specifically mention that when you are doing metagenomics, you also need to be very careful that you have a very "even" extraction protocol, so that the sequencing accurately reflects the sample's contents, and is not confounded by the extraction protocol that you are using.
7. Is VolTRAX commercially available?
Yes, VolTRAX is available in the Store (https://store.nanoporetech.com).
8. Which kit should I use to prepare a DNA library with the VolTRAX device?
The different VolTRAX kits are available in the Store (https://store.nanoporetech.com), and more will be released soon.
9. What is the difference between the flow cell R9.4 and the flow cell R10.3?
The difference is in the nanopores present in each flow cell. The nanopore is essentially a biological protein; it is found naturally occurring in the environment. We perform a lot of engineering internally to optimise these nanopores for utilisation in our system, enabling greater throughput and accuracy. The R9.4.1 nanopore is the current broadly-used nanopore. As DNA moves through the nanopore, there's a pinch-point - a narrowing of the hole, which allows us to measure the current, and so interpret the translocation of the molecule in real time. In the R10 series, there is a longer barrel and two pinch points, enabling two such measurements. This provides benefits for the sequencing of homopolymers - stretches of the same base multiple times. These homopolymer stretches are difficult, not only for our platform, but for all sequencing plaforms. We have found that introducing this second pinch point provides better fidelity for homopolymer sequences than previously seen with the R9.4 series; we are starting to see very accurate sequencing of homopolymer stretches of about ten bases. R10.3 is the newest nanopore in this series, and provides the high accuracy of the R10 series together with increased throughput and capture.
10. Can you please tell us more about library constuction using the Cas9 protocol? And for which applications it could be used?
The Cas9 PCR-free enrichment protocol has been available for about a year now; the protocol can be viewed in the Protocol Library in the Community. It is a fairly simple protocol; it takes about 2 hours, but we are making it even simpler, consolidating some of the third-party reagents that you need - this will be available very soon as a full kit. We have a few publications and videos about the use of Cas9 enrichment from our user community that are available online in our Resource Centre.
11. How does the accuracy vary throughout the run? Is the accuracy stable or does it decrease throughout the run, like for short-read sequencing technology?
Our technology can directly sequence native DNA; there is no need for amplification, enzymatic processes, fluorescence or anything else. We are just measuring the ionic flow as molecules pass through the pore. Because of this, and the fact that we can sequence very long molecules in single reads, the quality of sequencing is the same throughout the run; we don't see a decrease. As a DNA or RNA molecule passes through a nanopore, it is sequenced in its entirety, with the same fidelity at the beginning and end of the molecule. We also don't know what the upper limit is in terms of how long a molecule we can sequence in one read; the record is currently 2.44 Mb.
12. Could you talk about SNP detection with nanopore sequencing?
There are many different types of algorithms out there - both from the Community, and those provided by our data analysis team through Metrichor. One of these is the Medaka software package, which enables customers to perform different levels of SNP analysis. We are able to perform SNP detection - doing this in conjunction with the consensus strategy that I described will probably give the highest level of accuracy, and the ability to identify the different SNPs within the genomes you're looking at. We have seen quite a few publications over the last 18 months or so showing the accuracy of nanopore sequencing for the identification of SNPs in consensus sequencing; some very recent publications on SARS-CoV-2 sequencing show perfect concordance with other methods. These are available in the Resource Centre on our website; you can also always reach out to us at firstname.lastname@example.org.
13. For short genomes like plant viruses (10 kb), how many samples can I multiplex using a single flow cell?
A flongle Flow Cell will yield about 1 Gb of data, therefore it would be the equivalent of 100,000 reads 10 kb in length - often it is referred to as "100,000 X coverage". A typical MinION Flow Cell, where you could barcode up to 24 viral genomes, would yield likely over 20 Gb, or roughly 100,000 reads per barcoded sample.
14. To detect non-canonical methylations most of tools are trained based on well-known methylations. Some of the tools offer more flexibility but still require the use of a custom methylated sample as a reference for the detection of non-canonical methylations. Do we have any tools to just extract aberrant signals easily, assuming that these aberrant signals are from methylation?
Multiple tools exists to identify non canonical changes in the raw electrical signal. There are a number of publications and tools available in the Resource Centre which have used such an approach as the one that you describe. We also encourage you to get in touch if you have specific requirements.
15. Is the price on your website the price of the entire starter package or the price of renting the device?
If you are referring to the MinION, the price is the total price of the Starter pack, including MinION, or $1,000.
16. In WIMP, which databases are used for the taxonomy of the microorganisms?
WIMP is based on the Centrifuge classification engine.
17. If the platform is portable, does this mean that we can do sequencing when travelling with the device?
The sequencer has been used in space, cars, aeroplanes and even on a bike! See https://twitter.com/ReindertN/status/1235202393467428864
18. How stable is the Flongle Flow Cell with repect to the MinION Flow Cell?
We have been manufacturing MinION Flow Cells for a few years now. We warranty them for 3 months at delivery at 4 C, or 1 month at room temperature. Flongle Flow Cells at the moment need to be used within 4 weeks of delivery as per: https://store.nanoporetech.com/flongle-flow-cell-pack.html
20. Is it possible to perform direct RNA sequencing of plants in the field? Which extraction and VolTRAX library kits should I use?
Yes, it is possible to sequence directly in the field. We will have a follow-up Oxford Nanopore seminar on Extraction. Meanwhile, for more information on VolTRAX kits available, please visit the Sample Prep section of the Store (https://store.nanoporetech.com).
21. Could you explain how "Adaptive sequencing" works? Could it be used to sequence a family of genes, for example?
Absolutely! Please visit this page: https://nanoporetech.com/about-us/news/towards-real-time-targeting-enrichment-or-other-sampling-nanopore-sequencing-devices
22. Concerning VolTRAX versus Oxford Nanopore "hands-on" protocols, do they produce the same quality of libraries (length, distribution,...), and the same sequencing results?
Our internal tests show great concordance, but VolTRAX improves the inter-library reproducibility.