Long-Read Sequencing
Technologies
(Oxford Nanopore)

 

Results, guarantee and data analysis

Once the sequencing run is finished, you will receive its standard outputs from us. Because each sequencing project has specific data analysis requirements, we offer turnkey data analysis for the needs of your project. Our bioinformaticians can help you with any data analysis goal. If you are interested in this service, individual consultation is required, we recommend you seek our help already at the project preparation stage.

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Below is information on a few selected applications. The technological spectrum of Nanopore sequencers is very extensive, in case of other applications do not hesitate to contact us.

 

When identifying some organisms, e.g. microorganisms in complex communities, amplicon sequencing based on Illumina short reads is insufficient and longer stretches of DNA must be sequenced. A typical example is the sequencing of the entire 16S rRNA gene, the ITS region, etc. Oxford Nanopore technology is extremely suitable for this approach.Technologically, this is similar to any amplicon sequencing. Special primers compatible with subsequent sequencing are used and samples are multiplexed into one sequencing library. We will arrange the entire process for you on a turnkey basis, including long-range PCR on the gDNA supplied by you.

An alternative approach is shotgun sequencing of the entire sample or metatranscriptomic analysis. If you are interested in these services, please contact us.

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Comparison of Illumina and Oxford Nanopore outputs (example): Illumina technology is capable to discriminate down to the Bifidobacterium genus level whereas by using Oxford Nanopore technology we can identify species, e.g. Bifidobacterium breve.

What target regions do we use for taxonomic analysis?

Various hypervariable gene regions can be used for sequencing taxonomic analysis. Selection of primer combinations that we use for the amplification of these regions is continuously expanded. You can find the current available primer sets in our Sample submission sheet.

You need some other primers and cannot see them in our list? - Get in touch!

How reliable are Oxford Nanopore's taxonomic analysis results?

Oxford Nanopore sequencing technology is sometimes considered less accurate. This is true to a certain extent, but it is true in the sense of "technology A is 99.99% accurate and technology B is 99.999%". In reality, this technology is very accurate, "accurate enough", and only some specific applications (eg genome assembly) benefit from combining ONT with Illumina technology. This is not the case for metagenomic analyses.

Along with the laboratory processing of samples by ONT technology, data analysis can be ordered at the same time, which uses appropriate databases and results in graphs of taxonomic frequencies of individual categories and individual samples. It may happen that well-annotated databases do not exist for a certain group of organisms or a "less common" target gene. In this case, please contact us.

 

ONT technology is absolutely crucial in the assembly of new genomes, a bioinformatics process in which long or short (or both) types of reads are assembled. It can be likened to putting together a puzzle, where long ONT fragments of DNA can be assembled into a complete DNA sequence much more easily simply because they are longer, just as a puzzle with smaller pieces is easier to put together.

 

 

This makes long-read sequencing the technology of choice for genome assembly. However, for a number of reasons, the ideal scenario is to combine long ONT reads with short Illumina reads. Read about genome assembly using data from Oxford Nanopore and Illumina technologies! Projects like this typically require individual consultation.

 

Transgenic organisms (and mice in particular) are a critical tool for in vivo modeling.

The transgenic technique, where an exogenous gene is inserted into the mouse genome by direct injection of DNA into the zygote, has enabled the creation of thousands of new transgenic lines. However, the technique is not without limitations. One of its biggest drawbacks is that transgene integration is a random event.

But without knowing where the integration takes place, the consequences of a given genetic modification cannot be fully predicted. Insertion of a transgene can disrupt the regulatory or coding region of a critical gene, often multiple copies of the transgene are inserted as concatemers, leading to copy number variability, or different sites at which the integration event occurs can result in mosaic patterns of transgene expression (position effect) . Thus, it is very important to know the exact site of transgene integration.

Next-generation sequencing (NGS)-based methods can be used to map insertion sites, but Illumina's short-reads cannot reliably serve this purpose.

In our laboratory, for example, we detected the location of a transgene on the mouse X chromosome in the number of 18 consecutive copies (and one incomplete), while the total length of the analyzed region was about 80 kilobases. Such an analysis is completely beyond the capabilities of any short-read technology:

 

 

 

However, Oxford Nanopore's long read technology not only allows the transgene(s) to be located, but simultaneously:

• It provides information about the number of copies of the transgene
• Provides direct evidence of transgene inversions
• Detects contaminating E. coli genomic DNA
• Validates the integrity of neighboring genes

Note: Similar to genome assembly, a combination of ONT technology with Illumina short reads may be a suitable solution depending on the specific scenario. Projects of this type typically require individual consultation.