Frequently Asked Questions About Our Products And Services

Genome sequencing

• De novo sequencing of fungal genomes
• De novo sequencing of higher eukaryotic genomes
• De novo sequencing of BACs, Viruses & Plasmids
• Re-sequencing of genomes

Transcriptome sequencing

• De novo transcriptome sequencing
• Expression profiling

Resequencing & Amplicons

• Ultra Deep Amplicon Sequencing
• Resequencing by Sequence Capture
• Exome Sequencing

Bioinformatics & Special Services

• Bioinformatic solutions
  like de novo assembly, mapping, transcriptome analysis, amplicon variance analysis ...
• Library Service
• Sample Preparation
• NGS Favourites

If you order online (INVIEW and NGSelect products) the lab address will be displayed during checkout.

If you received a PDF quote the lab address will be included there. In general we have two Eurofins NGS labs:

Eurofins Genomics
NGS-Laboratory
Anzinger Str. 7a
85560 Ebersberg
Germany

Eurofins Genomics Europe Sequencing GmbH
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Both next generation sequencing technologies of Roche (454) and Illumina share a common approach, in, that before sequencing itself, the sequencing library has to be generated via PCR amplification of the DNA templates.

In the case of Illumina HiSeq 2000 or MiSeq, preparation of the sequencing library is done by bridge PCR, while the sequencing is done by a technology referred to as cyclic reversible termination.

In the bridge PCR primers and the DNA template are immobilised to the 2-dimensional surface. The primers are designed to target the adaptors of the DNA fragments so that the fragments bind to primers in their neighbourhood. Within each PCR cycle the fragments build so called bridges and the following denaturation leaves single stranded templates anchored to the surface. The copies remain local and form dense clusters. To sequence the clusters a universal primer is hybridized to the adaptor sequence of the DNA fragments.

Sequencing of clusters generated via bridge PCR is done by a technique called cyclic reversible termination. Thereafter, the polymerase extension is performed with reversible terminators. These are deoxynucleotides carrying a fluorophore and a blocking group. The 4 nucleotides have different fluorophores attached. The polymerase incorporates just 1 labelled nucleotide as the blocking group terminates DNA synthesis. Unincorporated nucleotides are washed away and the array is imaged to determine the identity of the incorporated nucleotide. This is followed by a cleavage step which removes the blocking group and the fluorophore. The resulting 4-colour images are used to decode the sequence.

In the case of Illumina HiSeq and MiSeq sequencing we ship FASTQ files of each single read. Intensity values and image files are automatically deleted during the runs and therefore cannot be shipped.

Yes, we have reference customers for all our technologies. Please find a selection of all publications here.

If you need a reference for a specific application, please contact genseq-eu@eurofins.com.

Please send the FASTA or NCBI file or the exact reference to genseq-eu@eurofins.com or to your sales contact person.

Yes, we have templates ready for signature, or we can sign your company/institute agreement, after review.

Confidentiality is guaranteed as part of our quotation and general terms and conditions.

Yes, we are a service provider, and as such, we do not claim any intellectual property.

This involves sequencing of a DNA until a certain number of sequence information and therefore sequence coverage is reached.

Example: A 1-fold sequence coverage of a 3 Mb genome in size means 3 million base pairs are determined, or a 6-fold coverage means 18 million base pairs are determined.

For genome sequencing using the Illumina technology we recommend a coverage of 30-50-fold.

You will receive your date from our secure FTP server.

If required we can also ship data on DVDs, USB sticks, or hard disk drives (depending on the amount of data).

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

To provide you with high quality sequencing data, we need high quality total RNA.

To determine the concentration of your isolated total RNA, we recommend either spectrometric methods like the NanoDrop system or chip-based electrophoretical technologies like the Agilent 2100 Bioanalyzer. Integrity estimations can be done by traditional RNA gel electrophoresis or with the Agilent 2100 Bioanalyzer.

We only start to prepare the library if your RNA sample has passed our QC.

Typical bioinformatics services after transcriptome sequencing are:

• De novo assembly of transcriptomes
• BLAST analysis

Clustering is the bioinformatic process of grouping sequencing reads that display a defined similarity. In contrast to the mapping processes, this is independent of an available reference sequence.

Clustering of sequencing reads is often performed to

• compare expression profiles of different mRNA samples for transcriptome analysis
• group different reads from the analysis of amplicon

A de novo assembly is an assembly of sequencing reads without using supportive information derived from a related reference genome. Therefore, each de novo assembly might deliver new and other than expected information in comparison to sequence data from any related organism.

An advantage of the method is that genetic rearrangements or insertions/deletions can be identified quickly.

During a genome assembly, "contiguous sequences of nucleotide bases" (contigs) are built from the multi-alignment of highly similar single reads.

After the alignment step, multiple consensus sequences of all aligned or assembled reads are obtained which represent the contig sequences of a given genome or assembly. In contrast, a scaffold is an ordered set of contigs which are linked by sequences that were derived from the paired-end information of long jumping distance libraries or mate-pair libraries.

Scaffolds always consist of contigs separated by gaps. These gaps might be identified by "NNNN" in a consensus sequence.

In contrast to "de novo assembly" a "mapped assembly" is a strictly reference-guided process that comprises the comparison and derived mapping of sequence reads with a reference sequence.

If reads are found to be similar to a reference region, they are mapped on it. If reads overlap with each other, contigs may be generated as well. The mapping approach allows the study of mutation positions (e.g. SNPs) or structural variations.

Yes, we offer both BLASTx and BLASTn alignments against the latest protein and nucleotide database releases.

BLASTn translates the DNA sequence in all possible reading frames and compares it with the non redundant NCBI protein database. BLASTx is a comparison of the DNA sequence with a nucleotide database of choice.