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Microbiology with Oxford Nanopore Technologies Q&As

Here you can find all of the questions and answers asked during the Microbiology with Oxford Nanopore Technologies webinar. 

1. Are there also workflows for strain identification available?

There are no readily available workflows. However, we have implemented it in our software COMMANDEr, and it is available for licensing.

2. Is there any software or tools to do variant calling from polyploid samples?    

There are no readily available workflows. However, we have implemented it in our software COMMANDEr, and it is available for licensing.

3. Are there recombination events in HPV serotypes? If so, how do you detect them?    

None reported thus far. If the recombination event happens in the target L1 region we are sequencing, we will be able to detect it. This is done by identifying reads which do not show complete alignment to the target region and have a significant stretch of sequence in a read mapping to another region.

4. What is the maximum number of barcodes Oxford Nanopore provides in their kits, to pool different samples?    

This is application dependent; up to 96 native barcodes are available for the ligation library prep, as well as two expansion packs of 12 different native barcodes. In PCR-based preps, you can use the 96 PCR barcode expansion pack, or choose to combine different barcoding strategies to multiplex much higher.

5. Is there a capture-based nanopore assay, not just a PCR-based targeted sequencing method?    

Yes, you have options of both CRISPR/Cas9-based capture and bait capture methods for targeted capture. "Digital capture" aka. adaptive sampling is also being worked on and is available as a research tool, under limited support.

6. What is the proportion of chimeric reads, due to concatamerisation, that you get during the barcode ligation process? How can we get rid of these?

We do see chimeric reads in the amplicon reads when we sequence with Oxford Nanopore. Specifically to this assay, it does not matter if there are chimeric reads, because we are just performing alignment. Is there a way to reduce the number of chimeric reads? Potentially one could improve on the ligation process; I think sometimes there is an excess of ligase we use in the library preparation protocol which could potentially lead to chimera formation, and I think that can be finetuned. Dan: The barcodes should be in massive excess and if they're not then I'd point you towards making sure that your end prep and sample cleanup work particularly well, and ensuring you have the appropriate amount of DNA, and quantify this accurately. Another possible cause could be in silico chimeric reads, perhaps if there's too much sequencing going on. If you're seeing it quite a lot I'd suggest using Porechop which will pull them apart for you.

7. Is it possible to sequence viral, bacterial and fungal genomes from the same sample, in the same run?

Yes, part of the library prep process is really just putting adapters on every free DNA molecule you have in solution, and it doesn't really discriminate as to what kind of DNA it is; of course if it's an RNA virus you will need to apply a different extraction and library prep protocol because those are different for RNA or cDNA vs. DNA. The analysis part might be slightly more complex but that is certainly doable as well. Sudha: Yes, it's absolutely possible to combine all kinds of samples. DNA is DNA, whether it comes from humans or plants etc., and the only thing one needs to take care of is how you normalise the libraries. Depending on how many reads you have, that can sometimes be a challenge because it's hard to predict the performance of the libraries.

8. Where can we find workflows and tutorials to analyse microbial data using nanopore sequencing? Is it possible to use the same tools used to analyse data generated from second generation sequencing technology?

If you register on our website for the online Community you will get access automatically as a customer. If not, you can register as a guest. With this, you can access the majority of materials, protocol builders, protocols, advice, and Community discussions etc. I would also point you to the Tools section of the Resource Centre on our website, to browse a wide range of analysis tools available for nanopore sequence data. Using the same tools as those developed with short-read data is typically not recommended as they can be an unknown beast; we always recommend using nanopore-specific analysis tools for nanopore data. Sudha: I think the tools are different because the read lengths are different and the chemistries are different, so one should use methods designed for long-read analysis. At least at the point of alignment. The more downstream analyses can be the same.

9. What level of training do you require for the operators of your mobile testing units?

I think one would need basic training in molecular biology; with a two-day training one should be able to get started with mobile testing units.

10. Have you drawn any conclusions on HPV variants based on the data that you have presented?

Whether or not we're able to predict the pathogenicity of the virus from the 16S variants is still a work in progess. In some populations they have identified specific genotypes that can be pathogenic, and we're just now building the data for the Indian population. I think the analysis interpretations will become finer as we get more data from our samples.

11. When bacterial diversity studies are performed, using 16S analysis, what level of identification can be achieved?

There are several different approaches. With 16S analysis alone, you could get to strain specificity in a few cases. We see people going beyond that by doing full ribosomal RNA operon sequencing, so instead of only 16S, sequencing 5 kb regions. It depends which questions you are asking, and what answers you want, and then your experiment can be designed accordingly. Sudha: We've done quite a few metagenomic sequencing projects and we find that the 16S amplicon that nanopore sequencing uses is larger than typical short-read sequencing analysis - it's the entire 1.5 kb region - so it allows a slightly higher resolution of identification to the strain level than sequencing on short-read platforms.

12. What can be the reason of such a low mapping rate of the reads to the virus genome? Can it be improved?

One reason is the viral load itself in the sample(s) we have tested. Second is some amount of off-target amplification that these primers generate, and that can certainly be improved to be more specific towards the target.

13. How much percentage variation in COVID-19+ patient samples is intra-host, as opposed to known mutations/strains?

There will be known SARS-Cov-2 mutation rates and known mutations. If it has been seen before, there will be clusters. Sudha: There is a metric to say that "this is different from another strain", but basically they do what is known as a phylogenetic analysis, which looks at the genomic variants and how they cluster. There is a very nice online database which is called GISAID - a global consortium where there are thousands of genomes submitted and they help you identify how close or different the strain you have is from known strains so far.

14. Sudha, am I correct in thinking that you're looking at bringing this to the field as a mobile test station? Why not rather collect samples and pool them for analysis in a stationary lab?

The reason for doing it mobile is to reach, geographically, a larger area. Logistics can sometimes be expensive and hard in India. The summers here are like 40 degrees and you don't know how samples are collected or how they are sent. It's a lot easier to process them on site. 

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