There are pros and cons of every sequencing method. Sanger sequencing and Oxford Nanopore sequencing (ONT) are both methods for determining the sequence of nucleotides in a piece of DNA. Typically Sanger is consider the most accurate method for short-read sequencing and NGS is better for long-read sequencing.

Overall, the choice between Sanger sequencing and ONT will depend on the specific needs of the application. Both methods have their strengths and limitations, and the appropriate method will depend on factors such as the length and quality of the DNA sample, the desired level of accuracy, and the cost and availability of the necessary equipment.

It really depends on the sample quality. We cannot guarantee the level of coverage at this time. A good sample submitted properly will typically yields hundreds or even thousands of sequencing reads. Consensus coverage depends on how many reads are full-length plasmids and how many, if any, degraded.

A coverage exceeding approximately 20x indicates a highly accurate consensus sequence.

A coverage below 20x can include inaccurate assembled plasmid sequences. The most common reasons for low quality coverage assemblies are listed in ‘Troubleshooting guide for failed samples’.

Any number of samples is welcome. There are no minimums or limitations.

You can send it and we can sequence it, but we cannot predict or promise the analysis outcome. The customer would take on the risk of these orders.

Great news! Yes we do. All information about our prepaid solution can be found here >>

Unfortunately not. Currently, we can only accept plasmids. However, we do offer a special service for amplicons and large DNA inserts, which is available for your use. Learn more >>

Unfortunately not. This barcode is required for our internal lab processes and is essential in ensuring that we can process your samples as efficiently as possible. Thank you for your cooperation!

Yes of course.

If you don't know if there is a dropbox close by, use our dropbox location page to find out.Please click here >>

Yes, FASTQ files are delivered for every project.

There are several reasons why your sample might have failed:

1. The starting material did not consist of circular DNA.
   In our library preparation protocol, circular DNA is required as the starting material. If linear DNA is provided, it will result in sample fragmentation and subsequently lead to poor or no results.
   
   
2. Insufficient DNA concentration in prepared samples.
   The primary reason for this issue is the use of a Nanodrop device to measure DNA concentration. We strongly recommend employing a Qubit or a similar method instead.
   
   
3. Presence of mixed plasmid species and/or fragmented genomic DNA or fragmented plasmids within the samples.
   You may detect signs of this failure mode through a wide range of read lengths reported in the raw read length histogram.
4. Contaminants in your samples like Ethanol. For more info please click here: https://community.nanoporetech.com/contaminants

To interpret your results you can also have a look at our troubleshooting guide. Click here >>

Please send your samples in nuclease-free water or elution buffer.

Please do not use a buffer containing EDTA.

For each run, we conduct multiple quality control (QC) measurements to assess the success of the library preparation and sequencing. If all the measurements indicate satisfactory results, we will not reprocess samples that have failed.

We will sequence your plasmids typically within 1 business day.

For plasmids > 25 kb please except a turnaround time of up to 5 business days.

The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.

So please handle these regions with special care.

Unfortunately not. The protocol used for the high-throughput Whole Plasmid Sequencing service does not allow single-stranded DNA.

But of course we can sequence your ssDNA molecules with our custom workflow. More information can be found here >>

• In order to offer our competitively priced product, we pool different samples on flow cell runs and re-use flow cells by washing them between runs. Both procedures are industry standard and performed according to the manufacturer’s specifications.  
• Barcoding and pooling samples on a single flow cell reduces cost and maximizes usage efficiency of the pores. However, nanopore sequencing has a higher error rate compared to “traditional” short-read next-generation sequencing, which can lead to misreadings in the barcode sequence and incorrect sample assignment/de-multiplexing at a rate of up to 0.05%. This issue is known as index hopping. 
• The flow cell washing procedure is very effective with only up to 0.1% of any previously loaded sample contaminating a subsequent run (known as carry-over contamination), nevertheless you might experience individual contaminating reads within your raw data.
• However, for both index hopping and carry-over it is important to note that a contamination of this level is unlikely to impact the final sequence assembly as several fine-tuned sequence cleaning and filtering steps are applied, provided your sample meets our quality and quantity standards and therefore yields sufficient numbers of high-quality plasmid reads. It is important to note, that the issue is most prominent in low coverage assemblies (see ‘What is the coverage?’).
• In the case that customer samples do not adhere to our sample specifications, the samples are sequenced on the customer’s own risk, and we cannot assume any liability for potential damages caused.
• If you’re concerned about contaminations and most importantly about reads from your samples appearing in other customer’s raw data, we offer the possibility to use our premium ONT services which include usage of a dedicated full flow cell for individual customers (learn more >>).

Here are a few kits:

VWR Plasmid Miniprep Kit II

QIAGEN Plasmid Plus Midi Kit 

peqGOLD Plasmid Miniprep Kit II

ZymoPURE II Pure Midiprep Kit 

For really large plasmids different kits are required. For example ZymoPUREII or NucleoBond.

On average, our turnaround time for bacterial genome sequencing, for genomes up to 7 Mb, is 3 to 5 business days.

Unfortunately not. This barcode is required for our internal lab processes and is essential in ensuring that we can process your samples as efficiently as possible. Thank you for your cooperation!

Yes of course.

If you don't know if there is a dropbox close by, use our dropbox location page to find out. Please click here >>

There are several reasons why your results might be different than what you expect.

To interpret your results you can have a look at our troubleshooting guide. Click here >>

For each run, we conduct multiple quality control (QC) measurements to assess the success of the library preparation and sequencing. If all the measurements indicate satisfactory results, we will not reprocess samples that have failed.

According to the specifications provided by Oxford Nanopore for the v14 library prep chemistry and the R10.4.1 flowcells currently employed in bacterial genome sequencing, the raw read accuracy exceeds 99%. The final assembly's accuracy fluctuates based on coverage and data quality. Generally, higher coverage, implying more reads for consensus construction, tends to enhance result accuracy.

Successful sequencing is determined by attaining at least one of the following outcomes:

1. A well-constructed genome assembly.
2. The specified quantity of raw data (equivalent to about 30x genome coverage of an average bacterial genome).

Deliverables:

• Data analysis report in HTML format
• Raw Sequencing data
• Assembled genome in FASTA format
• Circular genome plot
• Annotated genome sequence of all contigs in GENBANK format.
• Annotated genome features in GFF3 format
• Annotated feature table, including the nucleotide sequence of each feature
• Nucleotide sequences of all gene sequences in FASTA format
• Amino acid sequences of all annotated protein sequences in FASTA format

This service is designed for a clonal population (single strains) of bacteria. While you can submit mixtures of different bacterial strains for sequencing, it comes with a level of uncertainty in the assembly outcome, and therefore, it is done at your own risk.

If you are interested in mixed samples, we recommend using any of our other services:

• Microbiome profiling
• 16S Full-length sequencing
• INVIEW Resequencing of bacteria
• Whole genome sequencing on GridION (Illumina data polishing possible)

If your genomic DNA (gDNA) extract includes native plasmid DNA, it is likely that you will receive sequencing reads for those plasmids. Generally, DNA fragments with a size of <1 kb are excluded during sequencing cleaning step, because short reads do not help for a contiguous assembly and might include artifact sequencing. However, small plasmid-sized reads are not filtered out during assembly, so there is a probability that they will contribute to their own plasmid assemblies in addition to the gDNA assembly.

Ultimately, the types of DNA in your sample that contribute to an assembly will depend on the overall sample quality, coverage, and the relative abundance or degradation of each type of DNA.

Yes of course. Please check our full Oxford Nanopore portfolio here >> or send us your request >>.

A contig is the result of the assembly process, consisting of contiguous DNA sequences/ sequencing reads and not interrupted by unknown regions.

Even though it appears 100% perfect, there is still a likelihood of having single nucleotide polymorphisms (SNPs) in your sequence, especially in regions with homopolymers where Oxford Nanopore technology tends to have a higher error rate. To achieve a truly flawless assembly, we recommend using additional Illumina data to polish your results. Please feel free to contact us for further assistance.

The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.

So please handle these regions with special care.

For DNA sample preparation it is sufficient to use a high quality column based DNA preparation kit. The better the DNA quality, the better the results.

• In order to offer our competitively priced product, we pool different samples on flow cell runs and re-use flow cells by washing them between runs. Both procedures are industry standard and performed according to the manufacturer’s specifications.  
• Barcoding and pooling samples on a single flow cell reduces cost and maximizes usage efficiency of the pores. However, nanopore sequencing has a higher error rate compared to “traditional” short-read next-generation sequencing, which can lead to misreadings in the barcode sequence and incorrect sample assignment/de-multiplexing at a rate of up to 0.05%. This issue is known as index hopping. 
• The flow cell washing procedure is very effective with only up to 0.1% of any previously loaded sample contaminating a subsequent run (known as carry-over contamination), nevertheless you might experience individual contaminating reads within your raw data.
• However, for both index hopping and carry-over it is important to note that a contamination of this level is unlikely to impact the final sequence assembly as several fine-tuned sequence cleaning and filtering steps are applied, provided your sample meets our quality and quantity standards and therefore yields sufficient numbers of high-quality amplicon reads. It is important to note, that the issue is most prominent in low coverage assemblies (see ‘What is the coverage?’).
• In the case that customer samples do not adhere to our sample specifications, the samples are sequenced on the customer’s own risk, and we cannot assume any liability for potential damages caused.
• If you’re concerned about contaminations and most importantly about reads from your samples appearing in other customer’s raw data, we offer the possibility to use our premium ONT services which include usage of a dedicated full flow cell for individual customers (learn more >>).

Unfortunately not. This barcode is required for our internal lab processes and is essential in ensuring that we can process your samples as efficiently as possible. Thank you for your cooperation!

Yes of course.

If you don't know if there is a dropbox close by, use our dropbox location page to find out. Please click here >>

There are several reasons why your results might be different than what you expect.

To interpret your results you can have a look at our troubleshooting guide. Click here >>

For each run, we conduct multiple quality control (QC) measurements to assess the success of the library preparation and sequencing. If all the measurements indicate satisfactory results, we will not reprocess samples that have failed.

The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.

So please handle these regions with special care.

The assembler may encounter challenges reconstructing the terminal ends of linear DNA, leading to the potential omission of approximately ~10 nucleotides from the 3' and/or 5' ends of your insert, depending on your sample's sequence.

If you observe this issue with your samples, you can address it by downloading the raw reads from your Dashboard and reconstructing the ends using your preferred method.

We do not provide a specific coverage guarantee. The number of raw reads generated can significantly vary based on sample quality. Typically, successful samples sent at the required concentration yield a range from high dozens to hundreds (or even thousands) of raw sequencing reads.

The average coverage of your sample is included in the result report HTML, where a coverage exceeding ~20x indicates a very accurate consensus.

Our amplicon service is designed for a population of molecules that is clonal.

What will happen if you send mixtures?

If your species are sufficiently distinct (e.g. vastly different in size or sequence), the pipeline will first attempt to make a .fasta consensus file from the highest abundance species. It will also attempt to make a consensus of other species with read counts that are >20% of that of the most abundant species. Ultimately, which species end up producing a consensus will vary depending on overall sample quality, coverage, and relative abundance/degradation of each species.

How can I sequence my amplicon mixture?

Eurofins Genomics offers a vast amplicon sequencing portfolio. You will definitely find the correct service for your needs: Amplicon portfolio>>

• In order to offer our competitively priced product, we pool different samples on flow cell runs and re-use flow cells by washing them between runs. Both procedures are industry standard and performed according to the manufacturer’s specifications.  
• Barcoding and pooling samples on a single flow cell reduces cost and maximizes usage efficiency of the pores. However, nanopore sequencing has a higher error rate compared to “traditional” short-read next-generation sequencing, which can lead to misreadings in the barcode sequence and incorrect sample assignment/de-multiplexing at a rate of up to 0.05%. This issue is known as index hopping. 
• The flow cell washing procedure is very effective with only up to 0.1% of any previously loaded sample contaminating a subsequent run (known as carry-over contamination), nevertheless you might experience individual contaminating reads within your raw data.
• However, for both index hopping and carry-over it is important to note that a contamination of this level is unlikely to impact the final sequence assembly as several fine-tuned sequence cleaning and filtering steps are applied, provided your sample meets our quality and quantity standards and therefore yields sufficient numbers of high-quality amplicon reads. It is important to note, that the issue is most prominent in low coverage assemblies (see ‘What is the coverage?’).
• In the case that customer samples do not adhere to our sample specifications, the samples are sequenced on the customer’s own risk, and we cannot assume any liability for potential damages caused.
• If you’re concerned about contaminations and most importantly about reads from your samples appearing in other customer’s raw data, we offer the possibility to use our premium ONT services which include usage of a dedicated full flow cell for individual customers (learn more >>).

Unfortunately not. The protocol used for the high-throughput Clonal Amplicon Sequencing service does not allow single-stranded DNA.

But of course we can sequence your ssDNA molecules with our custom workflow. More information can be found here >>

It really depends on the sample quality. We cannot guarantee the level of coverage at this time. A good sample submitted properly will typically yields hundreds or even thousands of sequencing reads. Consensus coverage depends on how many reads are full-length amplicons and how many, if any, degraded.

A coverage exceeding approximately 20x indicates a highly accurate consensus sequence.

A coverage below 20x can include inaccurate assembled amplicon sequences. The most common reasons for low quality coverage assemblies are listed in ‘Troubleshooting guide for failed samples’.

We recommend purifying your amplicons with commercially available kits based on DNA-binding beads or columns or enzymatic cleanup. DO NOT ship any primers with your samples or mixed into your samples.

Oxford Nanopore Technologies (ONT) indeed allows for sequencing long reads, potentially up to several hundred kilobases. This depends highly on sample quality and DNA fragment lengths. However, to have a very competitively priced product we us a library preparation method which includes the adapters with minimal fragmentation (see examples in the troubleshooting guide). The resulting reads will not have the full-length amplicon size, but rather with a long-tailed size distribution.

For a full-length amplicon library preparation with adapter ligation the “premium” amplicon sequencing service>> can be used.

The term "amplicon assembly" refers to the process of reconstructing the original sequence from the sequencing reads (see above). The assembly process involves aligning and merging overlapping reads to create the assembled amplicon consensus sequence.

Read length distribution depends on sample quality, putative contaminants (see e.g. https://community.nanoporetech.com/contaminants), and for the amplicon product of course on the PCR specificity. While an agarose gel is definitely a good way to check the result of the PCR it lacks in sensitivity to detect bands with a low abundance (e.g. in comparison to electropherograms of a Bioanalyzer or Fragment Analyzer). These are usually the main reasons of “abnormal” read lengths distributions.

Most of the samples have read lengths cut off at the assembled size, which is a good indication that the full-length amplicon was assembled (and they also have coverage plots with an even read depth/coverage)