Frequently Asked Questions - Next Generation Sequencing

Yes we conduct a thorough entry quality control of your samples. Nevertheless, ensure to do your own quality control before you ship the samples in order to avoid delays or other pitfalls in processing.

• Samples are stored for 3 months
• Custom NGS primer are stored for 2 years

This is rather difficult to answer by general means, best you contact us to discuss your project in detail.

Please fill all wells column-wise.
For projects comprising more than 4 x 96-well plates please get in touch with us to discuss an efficient plate design.

Both amplicon and whole genome metagenome sequencing have their own advantages and disadvantages. Your method of choice should first be selected based on your research aim and second on given limitations. Benefits of Amplicon Metagenomics are:  high sensitivity, great assessment of the taxonomic composition and diversity, suitability and hence statistic power for a large number of samples. Benefits of Whole Genome Metagenomics are: Free of a taxon specific PCR-bias (nor restriction to a primerset dependent amplification).

Ribosomal RNA (rRNA) is the most abundant component (> 80%) in total RNA isolated from cells or tissues. However, for most applications, rRNA is irrelevant. Hence. its sequencing increases the required sequencing capacity and, ultimately, the costs of the analysis. If poly(A) enrichment is not possible or desired, total RNA needs to be ribo-depleted in most cases. Ribo-depletion further allows the detection of other RNA species than mRNA, therefore sequencing depth might need to be adjusted.

In most cases either due to species incompatibility (ribo-depletion is sequence and hence species dependent and sometime subtle sequence variations have a strong impact) of the sample and ribodepletion kit, or due to impurities in the sample (metabolites, high salt concentrations, etc.) or gDNA contamination.

To put it simple:

• Sequencing depth means sensitivity
• Read length means specificity
• Replicates mean confidence

Typical sequencing depths are 30 Mio read pairs (mammals, mRNASeq), 5 Mio read pairs (bacteria, mRNASeq; small RNA Seq), or 15-20 Mio read pairs (fungi, mRNASeq; ChIPSeq).

Samples with strongly degraded DNA or high concentrations of inhibiting metabolites are likely to fail in analysis or only provide a limited representation of the present species communities. To avoid false-positive results, a negative control (best: same – but analyte free – origin as sample) should be co-analysed with the samples.

Data is stored for 3 months.

Yes we do, please contact our bioinformatics specialists.

Beside resequencing many applications such as transcriptome sequencing rely on a high quality reference genome as basis for the deeper analysis. The full reference sequence ensures where to count, while the full reference annotation ensures what to count. Additional annotations of a reference genome allows additional deeper analysis such as pathway analysis, alternative transcript detection,…

There are always work arounds – please contact us for more information.

Depending on your choice of target regions, all bacterial, archaeal and fungal species of which a sequence is published in corresponding database (e.g. RDP, Unite, Silva) can be identified. There are currently for example thousands of species with sequence entries in the database. Samples are reported on species level, but closely related species with identical DNA sequences are not discriminated and are assigned to the best hit.

Choose at least one 16S target (for bacterial profiling), one ITS target (for fungal profiling) or customized, best peer accepted targets such as COI, rbcL, 12S or 18S. For Microsynths standard primer sets, please refer to our user guide Amplicon Metagenomics. Each primer set fails to amplify certain species, we recommend that you carefully check the literature available about the limitations of each primer set.

This could be due to several factors, e.g. the primer set selected might not amplify this particular species, the species can not be resolved fully as the amplified and detected sequence is too conserved (common for the whole genus or even a higher taxonomic level), the sequence is detected but not present in the database.