FAQs Sanger Sequencing

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.