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FAQs Sanger Sequencing

The right answers to frequently asked questions

Find the answers to all our products and services by clicking the links below.



What is the turnaround time for bacterial genome sequencing?

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

Can I send my samples without the free barcode?

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!

Can I use my local dropbox to ship samples for Bacterial Genome Sequencing?

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 >>

Troubleshooting guide for results

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 >>

Will you repeat sequencing of my sample if the sequencing fails?

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.

What level of accuracy can be expected from the results of bacterial genome sequencing?

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.

What are the expected outcomes or deliverables for a successful bacterial genome sequencing?

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

Is it possible to sequence a mixture of bacterial species in one sample?


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)

Is it possible to sequence the native plasmids of my bacteria along with their genomic DNA (gDNA)?

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.

Can you also sequence genomes larger than 7 Mb or genomes that are linear or multichromosomal?

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

What is a contig?

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

When my genome assembly looks perfect, can I assume that everything is correct?

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.

How about addressing the errors or positions with low confidence that I discovered in a homopolymer region or at a Dam or Dcm methylation site?

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.

Which kind of kit should I use to extract the genomic DNA?

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.

Are contaminations between samples possible (index hopping or carry-over contamination)

  • 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 >>).

 

Contact Us

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E-Mail: support-eu@genomics.eurofinseu.com

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Please direct inquires about pricing and special project requests to your sales representative.
General questions: support-eu@genomics.eurofinseu.com

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