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Metagenomics through nanopore sensing Q&As

Here you can find all of the questions and answers asked during the Metagenomics through nanopore sensing webinar. 

Caitlin Singleton:

1. Are bioinformatic analyse softwares used for metagenomic analyses are free of charge or online, like Qiime2, muthor...?

Yes, it is possible to do all bioinformatic analyses using open source software - generally you need to install the software on a server.

2. Hi Caitilin, Is this HQ-MAG database publicly available?

The MAG database is online, however only accessible to reviewers during pre-publication. Upon publication both the raw data and MAGs will be fully available in NCBI.

3. Can you suggest/predict an appropriate read depth range required for abundance/functional anaysis (enviro samples), and can you suggest analyses pipelines that have been the most efficient to date?

It really depends on your environment and target organisms. Deeper sequencing is required to catch the rarer populations. I would recommend searching the literature for studies in environments similar to yours. A coverage of 20x or more for your target organism is a good start.

4. How come this nanopore avoids other genome contaminations?

Nanopore sequencing does not avoid contamination in metagenomics. It helps reduce contamination for some members due to the long reads and fewer contigs, but you still need to be aware of contamination in your MAGs.

Oxford Nanopore Technologies:

1. You have talked about methylation. Can we know which base is methylated with nanopore sequence data? Could you please let me know more about the protocol and bioinformatics workflows involved?

Yes, you can determine if a specific base in methylated. You can find more details on our website here. Regarding the analysis side of things, there is an EPI2ME Labs tutorial on base modification analysis (see nanoporetech.com/analyse), and a range of tools developed by Oxford Nanopore (see the Oxford Nanopore GitHub page) and the Community (see the Tools section of the Resource Centre).

2. How much multiplexing is possible with 16S sequencing?

We curently have up to 24 barcodes available for the 16S kit: 16S Barcoding Kit 1-24 (SQK-16S024). Please visit the Store page for more information: https://store.nanoporetech.com/us/catalog/product/view/id/384/s/16s-barcoding-kit-1-24/category/28/.

3. What is the significance of 1D and 2D libraries? I want to know if using a 1D library instead of 16S will enable whole-genome sequencing of metagenomic samples?

The Ligation Sequencing Kit (SQK-LSK109) is ideal for whole-genome sequencing of metagenomic samples, as well as single microbial isolates. This kit will enable sequencing of genomic DNA that is native (unamplified) or PCR amplified. The 16S kit will only generate a 1.5 kb amplicon of the 16S rRNA gene, and therefore does not provide a whole-genome sequencing approach; this kit uses ligase-free attachment of Rapid Sequencing Adapters, as opposed to ligation-based, like the Ligation Sequencing Kit. All DNA sequencing kits are 1D chemistry; 2D chemistry has been retired.

4. What about microbial single-cell amplified genome (SAGs) sequencing with nanopore?

We are not aware of any publications using our platform for SAGs. In principle, you could sequence from these samples with Nanopore, provided adequate template material is available.

5. Will the output of nanopore technology support the SILVA, Greengenes, and HOMD databases?

The EPI2ME WIMP workflow does not support these databases, but yes, researchers have used these databases for microbial analysis with nanopore sequencing data. Please see this publication as an example: Nygaard et al. (2020), Scientific Reports. 10, 3209. https://www.nature.com/articles/s41598-020-59771-0

6. How much DNA or RNA and what quality are needed for metagenomic or transcriptome analyses; some environments are extreme and amount of recovered nucleic acids is limited?

When using the Ligation Sequencing Kit (SQK-LSK109) the standard input material is 1 µg of gDNA. For the 16S kits or cDNA, as little as 100-200 fmol of shorter-fragment input such as amplicons or cDNA can be used. The Metagenomics Getting Started guide and cDNA Getting Started guide in our Resource Centre both provide a lot of advice regarding input and QC.

7. Is DNA quantity a limitation for nanopore sequencing of environmental samples?

Possibly, depending on your individual sample; please refer to the specific library preparation kits for the required minimum amount of starting material.

8. Is this HQ-MAG database publicly available?

The MAG database is online, however it is only accessible to reviewers during pre-publication. Upon publication both the raw data and MAGs will be fully available in NCBI.

9. Can you suggest/predict an appropriate read depth range required for abundance/functional analysis (environmental samples), and can you suggest analyses pipelines that have been the most efficient to date?

It really depends on your environment and target organisms. Deeper sequencing is required to catch the rarer populations. I would recommend searching the literature for studies in environments similar to yours. A coverage of 20x or more for your target organism is a good start. The Metagenomics Getting Started guide in the Resource Centre may help you here as a starting point regarding both depth and analysis tool recommendations (https://nanoporetech.com/resource-centre/guide/metagenomic-sequencing).

10. How does nanopore sequencing avoid other genome contamination?

At the analysis level, long reads and fewer contigs can help reduce contamination, but you still need to be aware of it in your assemblies. If you are looking for information on host genomic DNA depletion, please refer to the Metagenomics Getting Started guide for advice.

11. What about metavirome analysis using Nanopore? How efficient is it and what are the advantages of using Nanopore?

There are two recent examples of metagenomic approaches for SARS_Cov_2 (COVID-19) viral detection.
1.    SISPA from PHE (https://www.medrxiv.org/content/10.1101/2020.03.05.20032011v1).
2.    Dr. Charles Chiu's MSSPE enrichment protocol (https://www.nature.com/articles/s41564-019-0637-9.pdf?draft=collection). Dr. Chiu has released primers specifically for SARS_CoV_2 as part of his MSSPE enrichment protocol (https://twitter.com/cychiu98/status/1221341348218822657). Furthermore, you may be interested in a recent publication on environmental bacterial and viral metagenomics using nanopore sequencing (Kate Reddington et al.: https://nanoporetech.com/resource-centre/metagenomic-analysis-planktonic-riverine-microbial-consortia-using-nanopore). A range of examples can be found in our Resource Centre.

12. Has Nanopore been applied to 18S analysis for fungi?

Oxford Nanopore does not have a kit that targets the 18S gene, but there are several papers that you can find in the Resource Centre that describe work looking at this gene.

13. Is there software for creating simulated Nanopore metagenomic samples, in order to test bioinformatics pipelines with known samples before using true samples?

Not as far as we are aware, but there should be public datasets from the many publications that are available. The Zymo Mock Community standards are often used for benchmarking, and such datasets are available (e.g. Samuel Nicholls et al., 2019. GigaScience: https://nanoporetech.com/resource-centre/ultra-deep-long-read-nanopore-sequencing-mock-microbial-community-standards.)

14. Has anyone tried nanopore sequencing from fungal-specific PCR amplicons for species diversity analysis? I tried but in the results I got everything but not fungal species.

There are a few customers that have looked at this but I do not think that their data has been published. Please contact our support team, support@nanoporetech.com for assistance. Nanopore sequencing of the 18S gene and ITS regions has been performed and such work published (please see the Resource Centre for example publications).

15. Is this 16S rRNA approach with nanopore sequencing approved for diagnosis?

Oxford Nanopore Technologies products are currently for research use only.

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