Agrigenomics solutions with nanopore sequencing Q&A
Here you can find all of the answers (given directly form our guest speakers) to all of the questions asked during the Agrigenomics solutions with nanopore sequencing webinar.
Speaker: Sebastian Theuns
1. Could you please tell us about costs and pricing?
For pricing information, please inquire by email.
2. Do you require any enrichment or host depletion?
We do an enrichment step otherwise viral sequences would be overshadowed by the bacterial and host DNA present in the sample. To speed up our analysis, we have developed a new sampler which is performing this enrichment immediately on-site and which leads to more accurate results.
3. What kind of samples do you prefer for the analysis?
We have built experience in analysing faecal sample, respiratory samples, serum samples, and a variety of tissue samples.
4. Are you able to differentiate between different strains of the same virus/bacteria? If you can, how?
Yes, this can be done using a genetic reconstruction of the whole/partial genome. One prerequisite is to have enough sequencing depth to be able to accurately call SNPs.
5. When identifying small viruses (10 kb genomes), how many samples can be multiplexed?
It all depends on the amount of input material (pure viral DNA/RNA) that is available and the sequencing kits you are planning to use. With the Ligation Sequencing kit and PCR Barcoding kits there are now options available to multiplex >24 samples in one run.
Speaker: Diane Saunders
1. When working with fungal samples, how important is the extraction, and how difficult is it to maintain long reads?
The MARPLE diagnostics method focuses on the amplification and sequencing of a highly variable gene set, where genes are typically less than 2.5 kb. Hence long read lengths are not required.
2. Is the 242 amplicon PCR in a single reaction? Is it just standard PCR?
The PCR reactions have been multiplexed so the amplification of the 242 genes is carried out across 4 reactions using a standard PCR protocol.
3. How robust do you expect your gene set to be regarding new/unknown strains not included in your training set, and/or hybrid strains if they occur in nature?
We tested an additional 13 yellow rust isolates that represent known race groups and were not included in the gene selection step. All of these isolates could be defined correctly using this method. As the method focuses on sequence analysis of a highly variable gene set rather than specific SNPs or SSR markers it is able to pick up any previously unknown, genetically distinct races.
4. How can you detect novel strains at low levels in mixed samples?
In this method we only use a single lesion for analysis, and from other work we have shown that this contains a single genotype of the pathogen.
5. Can you estimate the cost of the process?
The cost has yet to be optimised and at this point it costs ~£100 per sample, but in the next phase we plan to reduce this to under £50 per sample, and of course further if possible.
6. Did you identify any new isolates in Ethiopia with the approach in the field?
The analysis that we did showed that there had been no significant change in the yellow rust population from the previous year.
7. Does sample preparation need any precautionary steps to prevent false reporting?
It is important that single lesions are used for analysis to ensure a single genotype is present.
You can find the publication associated with Diane's talk here.