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Frequently Asked Questions About Our Products And Services

Next Generation Sequencing

General & Technical Questions

Which next generation sequencing services does Eurofins Genomics offer?

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

How can I order your next generation sequencing service?

As most next generation sequencing projects are very individual, please ask for a personal quote

In close cooperation we will work out your sequencing project.

Where should I send my samples?

Please send your samples together with a copy of your signed project quotation to:

Eurofins Genomics
Anzinger Str. 7a
85560 Ebersberg

Where can I learn more about the various sequencing systems (Illumina, PacBio)?


You can find additional information about this new technique provided by Illumina Inc. at http://www.illumina.com/applications/detail/sequencing/dna_sequencing.ilmn.

PacBio RS:

You can find additional information about this technique provided by Roche Applied Science at http://www.pacificbiosciences.com/products/.

What is the principle of the Illumina sequencing technology?

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.

FAQ 9.1

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.

FAQ 9.2

When using Illumina HiSeq 2500 sequencing, how can you adjust the sequencing data output to my requirements?

Flow cells of the high-output-mode of the Hiseq 2500 instrument are physically subdivided into 8 channels. In 1 channel up to 180 million clusters can be sequenced from one or from both ends.

Given this huge data output, we often recommend pooling of several samples in one channel in order to save sequencing costs. Here we use molecular barcodes (stretches of specific nucleotides) to tag up to 12 unique samples in a single channel. This is appropriate for most applications, like mRNA sequencing or ChIP sequencing.

Can you sequence GC rich DNA or DNA with secondary structures or hairpin structures?

Yes. However, de novo assembly of genomes with high or low GC content remains challenging. In these cases, best assembly results are achieved by sequencing a combination of shotgun and long paired end libraries.

Just contact us for design of the best sequencing strategy

What kind of data do you deliver for Illumina HiSeq and MiSeq projects?

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.

Do you have references for next generation sequencing projects?

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.

How do I provide a reference sequence?

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

Can I get a confidentiality agreement (CDA/NDA)?

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.

Do I own intellectual property of my results?

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

What are NGS Favourites?

Our NGS Favourites are straightforward solutions for complex NGS projects. They are predesigned packages evolved out of the individual project setups from the vast array of custom request.

Learn more about the NGS Favourites here.

Questions on Genome Sequencing Services

How much sample material do you need?

2-5 µg is the required amount of genomic DNA for shotgun sequencing on Illumina HiSeq 2500 / MiSeq.

Additional sample material is needed for the preparation and sequencing of LJD libraries with jumping distances of 3 kb, 8 kb, 20 kb and 40 kb. In this case, 5 µg, 15 µg, 30 µg and 50 µg respectively of extra DNA should be provided.

If possible, we would encourage you to submit some more sample material to allow for more intensive QC and repetition in case of failure.

What does coverage sequencing mean?

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.

What is the delivery time?

It depends on the organism and your requirement. For the coverage sequencing of a bacterial genome like E. coli (4.6 Mb), the delivery time is just a few weeks.

How do you deliver the data?

Depending on the amount of data, we ship data DVDs, USB sticks, or hard disk drives.

In addition to that we offer download of the data from our secure FTP server.

How is the assembly and scaffolding process done?

The assembly process assembles single overlapping reads to larger contigs. In the first instance, the contigs are not ordered. Scaffolding means to determine the order and orientation of the contigs. It can be achieved for example by sequencing long jumping distance libraries on the Illumina instruments. This technology creates pairs of short sequence tags, which are originally positioned 3 kbp, 8 kbp, 20 kbp or 40 kbp apart from each other.

Scaffolding matches the paired ends to the contigs under consideration of the original distance and thereby brings the contigs into order.

What is a mate-pair library?

Mate-Pair library consists of approximately 150-300 bp fragments. They are composed of 2 DNA segments originally located 2-5 kb apart in the genome of interest. With a mate-pair library it is therefore possible to span gaps or repeats of up to 2-5 kb.

For library preparation genomic fragments are sheared and size selected for approx. 3.5 kbp fragments. The fragments are end labelled with biotin and circularised. The circularised molecules are sheared once more and the library is enriched for biotin labelled fragments. Sequencing these biotinylated fragments generates the desired 'outward-facing' paired reads, meaning they align to a reference sequence outward-facing from each other. These outward-facing reads align to the reference sequence with a gap size of 2-5 kbp.

The mate pair library has several restrictions, limiting the usefulness of the library for high quality scaffolding of contigs:

1) Inward facing reads: Unbiotinylated fragments that have not been successfully washed away will cause undesired inward facing reads (see Illumina’s mate pair sample preparation guide). These inward-facing reads are like shotgun paired end reads as they align to the reference with a smaller gap size of around 200-300 bp.

2) Genomic shearing of the circularized biotinylated molecules is a mechanically process and will produce fragments having the biotin label in the middle of the fragment, but also on all other positions of the fragment. When sequencing fragments having the biotin label (and thus the crossover) in the middle of the fragment the desired outward-facing reads will be generated. However, when sequencing fragments having the biotin and the crossover at the outer 100 bps of the fragment, one of the resulting reads will be a hybrid read. The hybrid read will contain sequence from one AND the other end of the original 3.5 kbp fragment and there is no information about the location of the changeover. These read pairs are worthless for the assembly and can even lead to misassemblies.

For high quality high-throughput scaffolding by Illumina sequencing Eurofins Genomics offers an alternative proprietary library, the so called long jumping distance libraries (LJD's) with jumping distances of 3 kbp, 8 kbp, 20 kbp or 40 kbp. Read more

What is a long jumping distance (LJD) library?

Eurofins Genomics has developed long jumping distance (LJD) libraries for high throughput scaffolding of contigs. The LJD library is adapted to the Illumina HiSeq 2000 or the Illumina MiSeq technology. While the mate-pair library displays jumping distances of 2 kbp to 5 kbp, LJD libraries do show much more precise distances, thus improving assembly quality. LJD libraries are being offered for 3kbp, 8 kbp, 20 kbp and even 40 kbp jumping distance.

In addition, other advantages argue for the LJD library:

1. Because of the differences in library generation the number of shotgun paired end reads is greatly reduced. Only about 1% of LJDs reads are shotgun paired-end reads.

2. Within the LJD library the sequences from one and the other end of the original fragment (3 kbp, 8 kbp, 20kbp or 40 kbp) are separated by an adaptor sequence. Hybrid reads, as present in mate-pair libraries (see explanation in the mate-pair FAQ) are therefore almost completely eliminated when using LJD libraries. The changeover is defined by the presence of the adaptor.

Questions on Sequencing of Small Genomes and Large Insert Constructs

What kind of sample material do you need for sequencing of large constructs like BACs, PACs, fosmids or cosmids?

Please provide us with E. coli glycerol or stab cultures containing the vector constructs.

Is it possible to return the remaining DNA of my BACs, PACs, fosmids or cosmids?

Sure. If requested, we can provide you with the remaining DNA. In this case we will have to charge for handling and shipping of the DNA.

How much sample material do you need for sequencing small genomes like organelle DNA, viruses or phages?

We are able to start with 100-200 ng of DNA. A slight contamination with genomic DNA is acceptable, as long as it is kept <10%. In any case, we recommend sending non-contaminated sample material as this will avoid contaminating sequencing reads.

If possible, we recommend sending between 5-10 µg of sample material. This means we can avoid the amplification step which we have to apply for smaller sample amounts.

What does coverage sequencing mean?

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 150 kb BAC in size means 150 thousand base pairs are determined, or a 6-fold coverage means 900 thousand base pairs are determined.


How many runs per BACs, PACs, fosmids or cosmids do you need?

It depends on the size of your BACs, PACs, fosmids or cosmids and the required coverage.
Example: A quarter segment of a single run is enough to sequence 24 multiplexed BACs with sizes of up to 150 kb at a 20-fold coverage.

Please refer to your individual quotation.

How many runs per organelle DNA, virus or phage genome do you need?

It depends on the size of your small genome to be sequenced and the required coverage.
Example: 24 genomes of a size of 150 kb can be sequenced in a quarter segment at a 20-fold coverage.

Please refer to your individual quotation.

How do you deliver the data?

Depending on the amount of data, you will receive DVDs, USB sticks or a hard disk drives.

In addition to that we offer download of the data from our secure FTP server.

Questions on Transcriptome Sequencing Services

What kind of transcriptome analysis service do you offer?

What kind of sample material do you need for transcriptome analysis service?

Please provide us with total RNA.

After an initial quality control step, we isolate polyA+ mRNA from each RNA sample. If requested, we also isolate total RNA from tissue.

How much total RNA do you need for the transcriptome analysis service?

It depends on the type of library to be prepared. Before starting the library preparation, we perform a quality control on each RNA sample. Typical amounts of total RNA for mRNA-Seq libraries are at least 5 µg of high quality total RNA per sample.

In addition Eurofins Genomics does offer you cDNA library protocols for samples with really small amounts of starting material are available, like clinical samples, sorted cells and others. If you need a service partner for such projects just contact us.

What kind of concentration and quality measurement method do you recommend?

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.

What is the delivery time?

The delivery time depends on the type and size of your project. A standard project usually takes 6-8 weeks.

Please refer to your individual quote for more information

How do you deliver the data?

Depending on the amount of data, you will receive DVDs, an USB stick, or hard disk drives.

In addition to that we offer download of the data from our secure FTP server.

Is it necessary to have a reference sequence for expression profiling?

Yes, we need a reference genome or reference transcriptome sequence to do the mapping of reads and subsequent comparison of expression levels.

What kind of bioinformatic analysis do you offer for transcriptome analysis service?

Typical bioinformatics services after transcriptome sequencing are:

Questions on Small RNA Sequencing Services

How much sample material do you need for small RNA sequencing?

Please provide us with 10-20 µg of total RNA of each sample to be analysed. The absolute minimum is 5-10 µg. However, we would encourage you to submit more sample material, if possible. It allows for more intensive QC and repetition in case of a failure.

How do you prepare small insert cDNA libraries for small RNA sequencing on HiSeq 2000?

First of all, small RNAs are isolated from total RNA by acrylamide gel purification. Thereafter, 3'- and 5'-adaptors are ligated to small RNAs and finally the sequencing library is generated by reverse transcription (RT)-PCR and PCR amplification.

Questions on Amplicon Sequencing (focus on 16S)

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.

Questions on Targeted Resequencing by Sequence Capture

How do I provide target regions for enrichment?

The entire target sequence has to be provided in FASTA or multiple FASTA format. For non-human samples the minimum target size is 200 bp. All human target samples of <200 bp will be extended to 200 bp on the basis of the human genome sequence (NCBI data at UCSC). Your target enrichment sequence may consist of a continuous region or it may consist of independent regions (e.g. exons). Total length of target sequence is given by the array layout chosen.

What is the delivery time?

It depends on the size of your individual project and the sequencing technology applied.
Please refer to your individual quote.

What kind of quality controls do you perform before starting the sequencing of enriched samples?

The sequence capture techniques (on array or in-solution) contain built-in control probes.
We perform quality control on an amplified pool with qPCR to ensure system performance.

Can I perform sequence capture on organisms other than human?

In principle customised arrays can be designed for any other organism where reference sequences are available.

Is it possible to enrich repetitive regions?

Repetitive regions are not covered by the NimbleGen enrichment. They can not be sequenced.
However, repetitive regions that are flanked by unique regions are captured and be sequenced.

How much sample material do you need for sequence capture?

For sequence capture you have to provide us with at least 5 µg genomic DNA. NimbleGen Sequence Capture methods do not rely on amplification before hybridisation. Therefore potential sources of bias are excluded.

What regions can be captured by Roche NimbleGen Sequence Capture methods?

The customer can specify any region of interest where reference sequences are available, e.g. large contiguous genomic regions, for instance, whole chromosomes or non-continuous regions like specific exons or whole exomes of interest.

Who owns the design of the Roche NimbleGen Sequence Capture arrays?

Designs created for Roche NimbleGen arrays are property of Roche NimbleGen.

Are the array design data stored for further experiments?

Each specific customer capture design will be stored at Roche NimbleGen. You may order the same design in the future without additional design fees.

Is it possible to reuse sequence capture arrays?

Roche NimbleGen Sequence Capture arrays can not be reused.

How is sequence capture in solution performed with NimbleGen products?

  1. The SeqCap EZ oligonucleotide pool is made against target regions of the genome.
  2. A standard shotgun sequencing library is created from genomic DNA.
  3. The sequencing library is hybridised to the SeqCap EZ oligonucleotide pool.
  4. Streptavidin beads are used to pull down the complex of capture oligonucleotides and genomic DNA fragments.
  5. Unbound fragments are removed by washing.
  6. The enriched fragment pool is amplified by PCR.
  7. The success of enrichment is measured by qPCR at control loci.
  8. The end product is a sequencing library enriched for target regions and ready for high throughput sequencing.

NimbleGen Protocol

What is the general workflow of a customer definded targeted resequencing project with Eurofins Genomics?

  1. The customer selects an established exome design or any desired genomic target regions for enrichment.
  2. We handle oligonucleotide design, production and data transfer.
  3. We enrich genomic samples and prepare the sequencing library.
    Enriched samples can be sequenced with the Illumina HiSeq 2500.
  4. Bioinformatic analysis includes mapping of targeted re-sequencing data to a reference sequence, SNP analysis, quality statistic, coverage overview and other statistical analysis.
    Specific requests can be addressed with a customised bioinformatics project.

Questions on Bioinformatic Services

Do I need special software for further analysis of my sequence data?

It depends on what you are looking for. If you want to analyse the raw data or the assembly files, specialised software is necessary.

Please do not hesitate to contact us for project discussion

What does clustering mean?

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 sequencing projects

What is a de novo assembly?

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.

What is the difference between a scaffold and a contig?

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.

What does mapping stand for?

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.

Do you offer BLASTx and BLASTn analysis?

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

What is the difference between BLASTx and BLASTn?

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.

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