FAQs Sanger Sequencing

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)