The right answers to frequently asked questions
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What kind of samples can be used?
A wide variety of (DNA) samples can be used for microbiome analysis, such as:
- Environmental samples isolated from all kinds of sources
- Human samples, such as swabs, faeces, lavage
- Food samples for quality control and pathogen detection in industrial settings like dairies, breweries, meat-processing and agricultural facilities
How much starting material is required?
For information on the quantity and concentration required for specific library preparation methods please refer to your quote or contact us. For optimum results, we generally require >10 ng of double-stranded, purified DNA for PCR amplification (the total amount depends on the proportion of microbial DNA in the sample). Moreover, the DNA has to be RNA-free with an OD 260/280 of >1.8 and OD 260/230 of >1.9.
What data will I receive?
Eurofins Genomics will deliver tables (tsv, txt) listing all species present in the analysed sample and the corresponding read counts. In addition, you will receive all of the raw sequencing data.
Operational Taxonomic Units (OTUs), which are classified by BLAST analysis against a curated RDP database, are represented in a table format, including taxonomic classification down to the species level. Only the best hits are considered when assigning OTUs.
A demo data report can be viewed here.
Why is the 16S rRNA gene used for bacterial classification?
The 16S rRNA gene is ubiquitous among all bacteria. Its relatively small size of approximately 1,500 bp and its alternating structure of conserved and hypervariable regions (V1-V9) make the 16S rRNA well suited for identification and phylogenetic analysis of microorganisms. Excellent results are achieved with the V1-V8 primer pair. The option of using the V1-V9 primer pair is also available, but discouraged due to unstable primer attachment to the much smaller V9 region.
What is the necessary coverage for microbiome analysis?
The actual required sequencing depth mainly depends on the desired sensitivity and the complexity of the sample. The current sequencing coverage is about 15,000 reads per run. When in doubt, we recommend determining the required depth of sequencing by performing a trial on a sub-set of samples. The same applies to the experimental setup for pooling up to 10 samples.
Which organisms can be detected?
Phylogenic characterisation and analysis of microbial communities can be performed for various organisms from complex and non-complex food, industrial, environmental and clinical samples. However, not all organisms can be fully resolved using the 16S rRNA gene. The genus Bacillus is one bacterium that cannot be separated through 16S rRNA gene sequencing alone. We can overcome such technical limitations with the use of complementary detection methods with our Customised Solutions service, which offers the same quality as our standardised services.
How do I choose between Inview Microbiome Profiling 2.0 with Illumina and Inview Microbiome High Specificity with Pacific Biosciences?
Both services benefit from optimised PCR conditions with extremely low chimera formation.
The 2 x 300 bp paired-end read mode makes Inview Microbiome Profiling 2.0 ideally suited for sequencing several hypervariable regions at once. Scalable sequencing coverage allows for high sequencing depth as needed. The use of the latest Illumina chemistry provides a high degree of sensitivity and allows for the detection of even very low amounts of taxa in a complex sample. Furthermore, multiplexing makes it possible to process a huge amount of samples in parallel and within in a short turnaround time.
Inview Microbiome High Specificity with PacBio is the method of choice for sequencing of the entire 16S rDNA at the highest possible taxonomic resolution. Long amplicon sequencing allows for a high degree of specificity down to the species level. The Reads of Insert approach results in high-quality data output, where the threshold can be defined beforehand. The high degree of sensitivity of the full 16S analysis with SMRT sequencing technology makes it possible to characterise the microbial community, even at low densities.
Where should I send my samples?
Eurofins Genomics Europe Sequencing GmbH