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Frequently Asked Questions About Our Products And Services

What coverage is recommended for amplicon sequencing?

It depends on the goal of your experiment:

  • To detect rare variants with a detection limit of 5%, you need 1000-fold coverage
  • If you would like to have a detection limit of 1%, you need 5000-fold coverage, and so on

What Species can be detected using Microbiome Profiling by NGS?

All bacterial species of which a sequence is published in the NCBI nucleotide database can be identified. There are currently thousands of bacterial species with sequence entries in the database. Closely related species with almost identical DNA sequences will not be discriminated and only the genus (or the family) will be reported.

Limitations of using Microbiota Profiling by NGS

  • Closely related species are not distinguishable because of sequence identity.
  • Degradation of bacterial DNA may occur in highly processed samples or samples with low pH - a species identification is therfore difficult and sometimes not possible.
  • The more starting material we receive, the higher the probability to get excellent sequencing results.

What is the level of false positives / negatives?

Samples with strongly degraded bacterial DNA will give the result “no species identification possible”. To avoid false-positive results, a negative control (water) is always co-analysed with the samples.

With NGS all bacterial DNA amplicons that were successfully amplified from the sample DNA will be detected. Due to the high sensitivity of the method, sequences with low presence in the sequence pool or sequences with unexpected sequence lengths have to be removed from the data analysis during the bioinformatical workflow in order to exclude false-positives due to PCR and/or sequencing errors. Species with a fraction below 0.5 % of all assigned reads are not reported.

What kind of samples can be analysed?

The classical barcoding by Sanger sequencing is limited to pure samples or mixtures of at most 2 species, whereas the NGS approach can be used for the analysis of unknown mixtures with unlimited species numbers.

Regarding starting material you can send ready-to-sequence amplicon pools, purified DNA or source material.

Source material:

Fresh, frozen, and processed (canned, in oil, cooked (e.g. in ready meals)) food and feed products can be analysed. Additionally, the analysis of environmental samples (soil, water, feces…) is also possible. Please be aware that in the case of food and feed samples processing steps and additional ingredients in different formulas/recipes may also have an influence on the quality (integrity) of the bacterial DNA. Especially acidic ingredients (like vinegar) will degrade DNA. Samples should always be sent cooled or kept frozen.

Minimum quantity: 10 g of sample

What is the delivery time for a typical amplicon sequencing project?

The delivery time for Illumina MiSeq amplicon sequencing is maximum 20-30 working days after arrival of the DNA and all necessary information.

In addition we also offer an Express service for faster delivery.

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