Frequently Asked Questions About Our Products And Services

Under the conditions listed below, only simplified test reports may be provided.

Subject: Simplified test reports according to DIN EN ISO 170252018, (Section 7.8)

Testing Laboratory (Accreditation): Eurofins Genomics Europe Applied Genomics GmbH (D-PL-13372, ) Eurofins Genomics Europe Sequencing GmbH (D-PL-17038)

For accredited examinations at these two companies, test results may only be reported in simplified form, i. Requirements of the standard DIN EN ISO 17025: 2018 (section 7.8) are not fully applicable to this type of transfer of results.

These results can therefore also be reported as short test reports, electronically generated result reports, Excel spreadsheets or as result files.

 

If you have any questions, please contact the Customer Care Team.

All raw data (FAST-Q files) as well as the analysed and assembled data can be downloaded via your secure Ecom online account.

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

All raw data (FAST-Q files) as well as the analysed and assembled data can be downloaded via your secure Ecom online account. A demo data report for a typical project can be found under here.

SARS-CoV-2 Total RNA Genome sequencing

• Type: total RNA
• Quantity: 100 ng per sample
• Concentration: min 5 ng/µl
• Dissolved in: RNase-, DNase- and protease-free molecular grade water
• Please find in our sample submission guide detailed information about required sample volumes and shipping instructions

Artic SARS-CoV-2 RNA-Seq

• Type: total RNA
• Quantity: When the viral RNA has been previously assessed using a qPCR assay, the CT value should be between 18–35. If the CT is between 12–15, then dilute the sample 100-fold in water; if it's between 15–18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.
• Purity: OD 260/280: 1.8 - 2.0; OD 260/230: 2.0 - 2.2; RIN or RQI value ≥ 8
• Dissolved in: RNase-, DNase- and protease-free molecular grade water
• Please find in our sample submission guidedetailed information about required sample volumes and shipping instructions

SARS-CoV-2 Genome Sequencing

We guarantee 5 million read pairs (+/- 10%) per package.

Artic SARS-CoV-2 RNA-Seq

We deliver on average 5 million read pairs (+/- 10%) per sample. If the viral load is good (see our starting material requirements), then this is more than sufficient to assembly the full genome sequence.

Artic SARS-CoV-2 RNA-Seq

The ARTIC PCR approach is reducing the number of undesired, off-target reads for the host. If you aim for a full virus genome sequence and no additional information go for the Artic SARS-CoV-2 product.

You will not receive any information for the host/patient from the collected sample.

SARS-CoV-2 Genome Sequencing

If you aim to receive contextual meta-transcriptomic patient/oral flora information in addition to the viral genome/sequence information use this product.

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

All raw data are analysed and converted into a vcf file, which is uploaded directly to QIAGEN’s web-based software platform. Log-in data to your account will be provided by Eurofins Genomics.
Furthermore, the alignment file (BAM), the SNP and InDel tables (vcf and tsv) and the Data Analysis Report (pdf) from Eurofins Genomics will be delivered in the formats indicated via your online account (see below).

Please note: Customers must accept the QIAGEN End-User License Agreement before access will be granted to the Variant Analysis product or to the results generated by this product.

The license grants you initial access to the analysis software for 6 months. After that period you have the option of renewing the license, but you may only purchase a renewal offline – please contact us.

All raw data as well as the analysed data can be downloaded via your secured online account.

For SNP calling in heterozygote organisms we recommend at least 30x coverage. A higher coverage will increase the confidence of SNP calling. Confidently discovering genetic variants in inhomogeneous samples, such as DNA extracted from normal and tumour cells, requires higher overall coverage. The amount of data needed to reach a certain coverage on the DNA of interest depends on the ratio of normal-to-tumour DNA. Please note that due to varying efficiency of the enrichment baits, the targeted regions are not covered evenly (see below).

PCR duplicate rates are directly correlated to the quality and amount of starting material provided. Library preparation with less than the recommended amount of DNA requires additional PCR cycles to generate enough material to load the sequencer. Hence the PCR duplicate rate is dependent on the sample and cannot be influenced by Eurofins Genomics.
Duplicates are not excluded from the calculation of the average on-target coverage. Based on our experience with the sequencing of human samples, we typically obtain PCR duplicate rates of approximately 5% for high-quality DNA.
If further bioinformatics are ordered (e.g., SNP identification), only one copy of the duplicate read pair is kept in the alignment. The rest are excluded from further analysis to prevent any bias.

For optimum results we require at least 100 ng double-stranded, purified, high-molecular-weight, RNA-free DNA (concentration ≥ 1 ng/µL; OD 260/280 ≥ 1.8; OD 260/230 ≥ 2.0). For DNA from FFPE samples we need at least 200 ng of DNA.

Contamination by DNA from other species (> 3%; especially from closely related species) might interfere with the enrichment of the human DNA and also reduces the total amount of human DNA in the provided sample. Whole-genome amplified (WGA) DNA or DNA from archived tissue, such as Formalin-Fixed Paraffin Embedded (FFPE) samples, is usually accepted. Please contact us if you need any details.

The fragmentation of genomic DNA according to the Agilent SureSelectXT protocol and subsequent downstream processing produces a Gaussian distribution of DNA fragments with different lengths. A small percentage of the resulting library fragments have an insert size below 250 bp. Sequencing those library fragments with 150 bp paired-end reads will generate partially overlapping reads that cover the same bases and hence create an artificial doubling of coverage at those positions. To assure accurate analyses, those bases are excluded from further downstream analyses, such as single-nucleotide variant and insertion and deletion detection. Furthermore, Eurofins Genomics is continuously optimising its protocols to minimise the number of overlapping bases.

When sequencing human samples in 150 bp paired-end mode, Eurofins Genomics typically achieves over 80% of base calls with a quality value higher than Q30 ( >99.9% accurate).

For samples that have successfully passed the initial quality check at Eurofins Genomics, we will guarantee the on-target output you have ordered. Average on-target coverage is calculated as the sum of the mapped bases at each target position, divided by the number of bases in the target (bases on-target / size of target region).

The target region corresponds to the bait coordinates of the Agilent SureSelectHuman All Exon V6 Kit: approx. 60 Mbp of exonic bases (>93% of the targets are covered with >20x, 90% of the genes are completely covered – all exons – 10% of the genes are partially covered)

Due to varying efficiency of the enrichment baits (e.g., GC / AT content), targeted regions are covered differently and the range and uniformity of coverage varies over the target region. Eurofins Genomics applies the most recent Agilent Human All Exon kit design (V6) with improved design algorithms to better capture difficult regions and provide superior coverage uniformity.

The quality and quantity of each incoming sample will be determined by appropriate methods. Further quality controls are performed at various steps of the process.

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

We accept tissue, cells and blood as starting material for INVIEW Twist Exome. Whole-genome amplified (WGA) DNA or DNA from archived tissue, such as Formalin-Fixed Paraffin Embedded (FFPE) samples, is usually accepted. Please contact us if you need any details.

Data output only applies for freshly isolated, unamplified and pure human DNA samples (minimum 200 ng). Please note that contamination by DNA of other species (> 3 %; especially from closely related organisms) reduces the total amount of human DNA in the provided sample and it might interfere with the enrichment of the exonic region.

For the best possible coverage (> 99%) of protein coding genes in the human genome, we are using a combination of two panels. The Twist Human Core Exome (33 Mb) and the Twist Human RefSeq Panel (3.6Mb).

The rate of PCR duplicates correlates notably with the amount and quality of the provided sample material. However, with our optimized and fully automated library preparation process with sample type dependent DNA input and limited number of PCR cycles we increase library complexity and deliver the lowest possible PCR duplicate rate out of the provided DNA sample.
In addition, due to the patterned flow cell used for HiSeq4000 and NovaSeq6000 sequencers, the rate of PCR duplicates may be increased by technical duplicates.

If you order Bio-IT analyses of your samples (e.g. SNP identification) only one copy of the duplicate read pair is kept in the alignment. The remaining duplicates are excluded from further analysis to prevent bias.

Eurofins Genomics applies the latest Twist Bioscience technology with its dsDNA probes and specific probe design algorithm for efficient and uniform capturing of all possible target regions. In addition, our library preparation process is optimized to minimize amplification and enrichment bias.

Nevertheless, there might be some hard-to-cover regions, due their biological nature (e.g. high  AT or GC content).

For human samples sequenced with 150 bp paired-end mode, Eurofins Genomics typically achieves over 95% of base calls with a quality value higher than Q30 ( >99.9 % accuracy).

The alignment file (bam), the SNP and InDel tables (vcf and tsv) and the Data Analysis Report (pdf) will be delivered in the given formats via your Ecom account.

Should you choose optional access to QIAGEN’s Ingenuity Variant Analysis platform, all raw data will be analysed and converted into a vcf file, and then directly uploaded to QIAGEN’s web-based software platform. Log-in data to your personal account will be provided by Eurofins Genomics.

 Please note! Customer must accept QIAGEN’s End User License Agreement before access is granted to Ingenuity Variant Analysis and any data generated from the service.

The license grants access to the analysis software for six months. After this period, an optional renewal of the license can be purchased offline exclusively – please contact us.

Both INVIEW Twist Exome and INVIEW Human Exome Advance are excellent one-stop solutions for whole exome sequencing. The products offer sequencing according to diagnostic standards (ISO17025) and comparable BioIT pipelines for data analysis. Both services also offer complementary data processing with QIAGEN’s Ingenuity Variant Analysis. The two products vary slightly in their workflows, applications and associated costs

INVIEW Twist Exome is the most cost-efficient solution for whole exome sequencing. The product is ideal for analysis of germline mutations.

INVIEW Human Exome Advance is a more flexible whole exome sequencing solution. Inview Exome Advance is offered with guaranteed average on target coverage in multiples of 30x. The product can be used for analysis of both germline and somatic mutations.

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

For INVIEW Oncopanel All-in-one Liquid Biopsy, we accept fresh blood (must be provided in BCT with a stabilising buffer intended for the isolation of cfDNA) or plasma samples or retained plasma samples as well as already isolated cell-free DNA. Please refer to our sample preparation sheet. The extraction of cfDNA is performed from the blood plasma fraction.
For starting material like DNA isolated from FFPE and fresh-frozen tissue samples, please refer to our product aimed at solid tumour profiling, INVIEW Oncopanel All-In-One.

We recommend the delivery of two 10 ml blood samples. The first 10 ml blood sample is used for testing, whereas the second sample can be stored as reserve material. For plasma samples the optimal amount is 4 ml. Please note, that with smaller plasma volumes the performance and sensitivity can decrease. Alternatively we accept 15 ng already isolated cell-free DNA (up to 30 µl / concentration > 0,5 ng/µl).

The genes of interests are enriched via proprietary Eurofins protocols using the latest Agilent SureSelect hybridisation technology. The target enrichment system captures genomic targets using long 120 nt RNA baits. The hybridisation-based strategy facilitates the deduction of PCR duplications in the assay. The Eurofins proprietary protocol enables the generation of highly complex libraries, deep coverage of regions of interest and improved sequence uniformity. Following target enrichment, next-generation sequencing of the library is performed.

The product interrogates the exons of about 600 genomic regions that are implicated in cancer development. Thereby, an extremely high number of possible mutations are covered and analysed. The detected genomic aberrations include SNPs, InDels, CNVs and gene fusion events.

SNP and InDel detection has a technical sensitivity down to 1%.

The quality and quantity of each sample will be determined at sample receipt or after DNA extraction. Further quality controls are performed at various steps of the process.

All three tests can be used with liquid biopsy samples (cfDNA). The three products serve as powerful non-invasive tools for cancer profiling.

INVIEW Oncoexome Liquid Biopsy is the first commercially available service for whole exome sequencing (WES) of cfDNA. With this service, a comprehensive profile of all mutations present in the protein coding regions of the exome is obtained. This product is suitable for a comprehensive look at the exome of a cancer patient, in cases where limited information regarding clinically relevant mutations is available.

INVIEW Oncopanel All-in-one Liquid Biopsy is a comprehensive NGS cancer panel for profiling important tumour-specific mutations implicated in nearly all cancer types. The service uses Agilent SureSelect with an optimized protocol to target about 600 known cancer genes including tumour suppressors, mutation hotspots and drug resistance markers. The panel is optimised for sequence coverage and uniformity with sensitivity down to 1%.

INVIEW Oncotarget facilitates ultra-sensitive detection of clinically relevant cancer-specific point mutations, insertions, deletions and gene fusions in circulating cell-free DNA down to an allele frequency of 0.1%. This approach is suitable for treatment monitoring as it has the appropriate sensitivity to detect a relapse at a very early stage.

The product is available for research use only (RUO). All samples are processed compliant to ISO 17025:2005 accreditation.

Even when control samples (plasma samples from at least seven other patients/individuals) are submitted, the CNV analysis approach could be subjected to limitations. The sensitivity and specificity of the assay will depend on the level of CNVs present and the tumour fraction.

Tumour heterogeneity could make it difficult to correctly identify all relevant copy number variations in a given plasma sample. In addition, excessive contamination with DNA from normal cells could lead to coverage differences that are too small to be detected even with Eurofins Genomics highly sensitive methods.

All deliverables (FAST-Q files and analyzed data) can be downloaded via your secure Ecom online account.

We recommend that the blood sample be shipped to Eurofins Genomics within 24 hours after blood drawing. The sample must be stored in BCT Streck tubes. Storage and delivery of blood samples must be at room temperature. Plasma samples have to be shipped on dry ice.

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

For the INVIEW Oncopanel, we accept fresh-frozen and FFPE tissue and genomic DNA.

For liquid biopsy analysis of cell-free DNA (cfDNA) isolated from blood plasma, please refer to our GATCLiquid Oncopanel All-In-One product.

The genes of interests are enriched via a proprietary Eurofins protocols. Target enrichment approach outcompetes common PCR-based enrichment with very uniform coverage and all exons of the genes covered. The proven target enrichment method has been optimised by Eurofins to develop highly complex libraries from low DNA and to deliver deep and uniform sequence coverage. Following target capture, next-generation sequencing of the library is performed.

The service fully covers the entire exons of about 600 cancer-relevant genomic regions, including protein-coding genes, select promoter regions, miRNAs, extra-exonic variants and select fusion gene events. In addition to SNPs and InDels, the product can also detect CNVs.

• TMB is defined as the number of somatic, coding, base substitution, and InDel mutations per megabase of genome examined. All base substitutions and InDels in the coding region of targeted genes, including synonymous mutations, are initially counted before filtering as outlined below.
• The following mutations are excluded in silico from the TMB computation: Known somatic mutations in COSMIC and ClinVar, low-confident mutations, known germline variants in the ExAC (gnomAD) database, mutations predicted to be germline by the somatic-germline-zygosity algorithm, and mutations in tumor suppressor genes due to the focus of the Oncopanel All-In-One on actionable cancer mutations and potential panel design bias.
• To calculate the TMB per megabase, the total number of mutations counted is normalized by the size of the coding region of the targeted region in megabase (mutations per megabase, mut/MB). Due to the lack of standardization in TMB quantification we provide three TMB values:
  • Non-synonymous mutations
  • Non-synonymous mutations and plus indels and iii) including all mutations.

Although the gold standard for TMB determination is whole-exome sequencing (WES), focused panels, like the Oncopanel All-In-One v2.0, have been evaluated extensively for TMB estimation because of their lower sequencing cost and higher sensitivity. Recent research shows, that panels with a size > 1.5 Mbp are sufficiently suited to determine TMB with a high concordance to WES (Buchhalter et al. 2019, Int J Cancer 144:848–858). Thus, Oncopanel All-In-One 2.0’s size of 3 Mbp provides more precise TMB estimates than other smaller panels.

SNP and InDel detection has a technical sensitivity down to 1%.   

The quality and quantity of each sample will be determined at sample receipt or after DNA extraction. Further quality controls are performed at various steps of the process.

Both tests offer cancer variant detection services based on a validated oncology panel. The interrogated genes and genomic alterations of the panel are the same. Similarly, the techniques used for both products are based on hybridisation for target enrichment and next-generation sequencing.

The two products differ in their starting material. Inview Oncopanel All-In-One can accept conventional source material like fresh-frozen and FFPE tissues, whereas GATCLiquid Oncopanel All-In-One analyses cfDNA extracted from blood plasma samples.

Yes, the two services are ideally suited for studies aimed at comparing the performance of traditional biopsies versus liquid biopsies.

Even when matched samples (tumour tissue and normal tissue or blood from the same patient) are submitted, the paired analysis approach could be subjected to limitations. The measurement variance on this single matched reference sample will be higher than that for a reference consisting of multiple reference samples. This could cause a modest number of (false-positive) CNV calls even in cases where the two paired samples are truly copy number identical.

Tumour heterogeneity could make it difficult to correctly identify all relevant copy number variations in a given sample. Even when the tumour itself is homogeneous, excessive contamination of normal cells within a tumour sample could lead to coverage differences that are too small to be detected even with Eurofins Genomics highly sensitive methods.

The product is available for research use only (RUO). The service is performed in an ISO17025:2005 –accredited and ISO13485:2016-certified laboratory.

All deliverables (FAST-Q files and analyzed data) can be downloaded via your secure online account.

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

For Inview Metagenome Advance we accept clinical research samples of human origin (e.g. swaps, feces, lavage). Upon request many types of starting material can be used for whole metagenome analysis such as:

food samples for quality control and pathogen detection in an industrial environment (dairies, breweries, meat-processing and agricultural facilities)
environmental samples
Eurofins Genomics is particularly well-experienced in DNA isolation and sequencing of stool samples.

For specific information on quantity and concentration requirements for specific library preparation methods please refer to your quote or contact us directly. For best results we typically require >100 ng of double-stranded, purified DNA (total amount depends on the proportion of microbial DNA in the sample). Please note that the DNA has to be RNA-free with an OD 260/280 >1.8 and OD 260/230 >2.0.

Eurofins Genomics will provide you with tables (tsv, txt) that list all genera present in your sample and the respective read counts. In addition, you will receive all raw sequencing data.
Operational Taxonomic Units (OTUs), which are classified by Kraken’s exact k-mer alignment algorithm and appropriate MiniKrakenDB database, are represented in table format with taxonomic identification.

The required sequencing depth for successful metagenome sequencing mainly depends on the complexity of the sample (number and representation of individual species) and the level of expected host contamination. Microbial communities of high complexity with high background levels of host DNA require more coverage than samples of reduced complexity and with lower host DNA contamination levels. In case of doubt we recommend performing a pilot on a sub-set of samples to determine the required sequencing coverage.

Yes, Inview Metagenome Advance comes with guaranteed 10 million read-pairs. Additional read packages can be ordered separately.

Metagenomics analysis permits the identification of microorganisms independent of taxonomic markers such as 16S rRNA. Metagenome sequencing enables the identification of both culturable and unculturable organisms, such as bacteria, archaea, fungi, protozoa and viruses. Although Eurofins Genomics is most experienced in profiling human microbiota, we have also successfully characterised microbial community members from food, industrial and environmental samples.

Both amplicon and metagenome sequencing present distinct advantages as well as disadvantages. Your method of choice should be based on your research goal. The successful outcome of a project typically depends on several factors such as community composition, the abundance of closely-related species and sequencing depth.

Amplicon sequencing offers suitable taxonomic profiling of a large number of samples. The approach enables the detection of subtle differences between microbial communities, which makes the method beneficial for statistical comparisons, such as for case control studies or for sampling varying environments over time.

Metagenomic sequencing does not rely on a certain set of primers, which frees the method of taxon-specific PCR biases. This makes possible more accurate representations of the analysed microbiota and more dependable estimations species abundance. Metagenomics analysis can provide information on both the taxonomic as well as the functional characteristics of a given sample.

Inview Metagenome Explore is a ideally suited for broad taxonomic profiling of all organisms present in a given microbiome. The included 10 million read pairs are suitable for analysis of low complexity metagenomes, as well as for a broad overview of genera in complex metagenomes. A deeper sequencing coverage can be accomplished by adding additional read packages.
Inview Metagenome Advance provides detailed assessment of antibiotic resistance and community function, in addition to taxonomic characterisation. The product is ideal for profiling of complex metagenomes. For a deeper sequencing coverage than the included 10 million read pairs additional read packages can be added. The service can be applied to the quantification of functional processes in a particular community or to the detection of numerous resistance factors in an undisturbed natural environment. Please note that the required sequencing depth for successful metagenomic profiling is strongly influenced by the complexity and expected level of host contamination of the starting material. Therefore, additional sequencing effort could be necessary to obtain satisfactory results.

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

In general all kind of (DNA) samples can be used for metagenome profiling such as:

• samples of human origin (swaps, feces, lavage) 
• food samples for quality control of microorganisms and pathogen detection in an industrial setting (dairies, breweries, meat-processing and agricultural facilities)
• environmental samples

Eurofins Genomics is particularly well-experienced in DNA isolation and sequencing of stool samples.

For information on quantity and concentration required for specific library preparation methods please refer to your quote or contact us. For optimal results we generally require >100 ng of double-stranded, purified DNA (total amount depends on the proportion of microbial DNA in the sample). Please note that the DNA has to be RNA-free with an OD 260/280 >1.8 and OD 260/230 >2.0.

Eurofins Genomics will deliver tables (tsv, txt) listing all genera present in the analysed sample and the respective read counts. In addition, you receive all raw sequencing data.
Operational Taxonomic Units (OTUs), which are classified by BLAST analysis against appropriate databases, are represented in a tabular form including taxonomic identification. Only the best hits are considered for OTU assignment.

The required sequencing depth for successful metagenome sequencing mainly depends on the complexity of the sample (number and representation of individual species) and the level of expected host contamination. Samples of high complexity with high background levels of host DNA require more coverage than samples of reduced complexity and with lower host DNA contamination levels. In case of doubt we recommend performing a pilot on a sub-set of samples to determine the required sequencing coverage.

Yes, Inview Metagenome Explore comes with guaranteed 10 million read-pairs. Additional read packages can be ordered separately.

Metagenomics analysis permits the identification of microorganisms independent of taxonomic genetic markers. The approach enables the identification of culturable and unculturable organisms, such as bacteria, archaea, viruses, as well as simple eukaryotes including fungi and protists. Although Eurofins Genomics has the most expertise in profiling human microbiota, we have also successfully identified microbial community members from food, industrial and environmental samples.

Both amplicon and metagenome sequencing have their own advantages and disadvantages.

Your method of choice should be selected based on your research aim. Generally, the successful outcome of a project depends on several factors such as community composition, the abundance of other close species or strains and sequencing coverage depth.

Amplicon sequencing offers suitable assessment of taxonomic composition and diversity of a large number of samples. As you have more power to detect subtle differences between microbial communities, the method is beneficial for statistical comparisons, such as for case control studies or for sampling environments over time.

Metagenomic sequencing is free of taxon-specific PCR biases due to the primer sets used. This enables more accurate representations of the analysed microbiota and more dependable estimations of species abundance levels. Metagenomics analysis can also help elucidate both taxonomic as well as functional characteristics of a given sample.

Inview Metagenome Explore is a great product for the general taxonomic profiling of all organisms present in a given microbiome. The included 10 million read pairs are suitable for analysis of low complexity metagenomes, as well as for a broad overview of genera in complex metagenomes. A deeper sequencing coverage can be accomplished by adding additional read packages.
Inview Metagenome Advance offers detailed assessment of antibiotic resistance and community function, in addition to taxonomic characterisation. The product is ideal for profiling of complex metagenomes. For a deeper sequencing coverage than the included 10 million read pairs additional read packages can be added. The service can help quantify functional processes in a particular community or detect numerous resistance factors in an undisturbed natural environment.

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

This depends on many factors, such as tissue type, sample quality, development status, existing references, etc. Please refer to the table shown under Product Specifications to find your optimum InView™ Transcriptome solution. The estimated read numbers shown there refer to human samples. Alternatively, please contact us to discuss your project. In some cases it might be advisable to set up a trial to define how many reads will be required for your specific aim or organism.

The INVIEW Transcriptome Explore package gives you an overview of the expressed genes and is well suited to detecting differences between samples.

INVIEW Transcriptome Discover uses a strand-specific RNA library combined with Illumina’s 150 bp paired-end sequencing and is therefore recommended for the detection of differentially expressed genes, rare and novel transcripts and for discovering splice variants. In addition, the information about the transcript’s orientation allows for a more precise determination of structure and gene expression.

RNA-Seq of bacterial samples most often requires less reads since no splice variants are present. The INVIEW Transcriptome Bacteria is a cost-alternative for all projects interested in the gene expression profile of Bacteria.

The INVIEW Transcriptome Ultra Low is specially designed for all projects with only a tiny amount of starting material.

INVIEW Transcriptome Discover:

Not necessarily. However, if you have a reference, please refer to the recommendations below.

INVIEW Transcriptome Explore / Bacteria / Ultra Low:

A clearly defined Ensembl name (e.g., GRCh37 or Rnor_5.0) for the annotated genomic reference sequence has to be provided prior to project start. Alternatively, the genome sequence can be provided in Fasta (along with the corresponding annotation in Gene transfer format (gtf)) or in GenBank format (including annotation). For projects without a reference genome, de novo transcriptome sequencing based on PacBio RS II technology can be added.

Total RNA from tissues, cells or body fluids from eukaryotic or prokaryotic organisms can be used to prepare libraries for sequencing. In addition to RNA isolated from fresh tissue, RNA from FFPE samples can be analysed. RNA isolation can be offered as an additional service. For starting material from other sources please contact us.

INVIEW Transcriptome Ultra Low:

Please note that we cannot accept cells in cell culture media for the INVIEW Transcriptome Ultra Low package.

For information on the quantity and concentration of RNA required for specific library preparation methods, please refer to your quote or contact us. In general, we require 150 ng of high-quality RNA (up to 25 µl / optimal concentration 6 - 50 ng/µl (absolute maximum concentration 200 ng/µl)) with an OD 260/280 of 1.8-2.0 and an OD 260/230 of 2.0-2.2 for optimum results. It might be possible to start with less material, but this depends a great deal on the quality of the material and the requested library.
The quality and quantity of the provided starting material are crucial for the success of sample preparation. The use of too little, degraded, contaminated or otherwise damaged starting material can result in a low yield or sample preparation failure and impair the quality and quantity of sequencing results.

INVIEW Transcriptome Ultra Low:

Minimum 0.15 ng/µl (in min. 10 µl); RIN > 6.5

Eurofins Genomics offers RNA isolation as an additional service. We have successfully optimised protocols for extraction of high-quality RNA from several sample types and starting materials. Please refer to Product Specifications for more information on RNA isolation or consult us directly to discuss possible sample preparation methods for your individual requirements.

a) For eukaryotes: rRNA depletion is recommended for partially degraded RNA or for when sequencing RNA with no poly-A tail.

b) For prokaryotes we recommend rRNA depletion in general, because poly-(A) enrichment of prokaryotic transcripts is not feasible.

You can order rRNA depletion from Eurofins Genomics. Or, alternatively, you can provide us with RNA that has already been rRNA depleted / mRNA enriched as starting material (20 ng per sample (up to 15 µl / concentration >1,4 ng/µl)). Please see Product Specifications for more information.

The efficiency of removal is determined by the organism, the composition and the quality of your sample RNA. If the RNA is severely degraded, ribodepletion might be less efficient.

RNA must be shipped on dry ice, unless the RNA is precipitated in ethanol. Tissues / cell cultures must be flash frozen in liquid nitrogen or dry ice and have to be shipped on dry ice. Alternatively, fresh material can be stabilised in “RNAlater” (e.g., Ambion, SIGMA or QIAGEN) and be sent at room temperature. Please verify in advance that the couriers you are using accept dry-ice shipments.

If the amount, concentration or quality of the starting material does not meet requirements for further processing, we will contact you to discuss how to proceed further. If possible, Eurofins Genomics will recommend additional pre-processing steps in order to optimise sample quality.

All raw data (FAST-Q files) as well as the analysed data can be downloaded via your secure online account.

Affordable high-quality bioinformatics analysis, including data filtering and QC, is optionally available for each package.

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

For a list of accepted starting material, including the relevant amounts, please refer to Table 1 in Product Specifications.

NGSelect Amplicon Adaptor Ligation:

Prior to sequencing, the quality and quantity of each sample will be determined by established methods (e.g. agarose gel analysis / Qubit® Fluorometer / NanoDrop / Agilent 2100 Bioanalyzer). Further quality controls are performed at various steps of the sequencing workflow.

NGSelect Amplicon 2nd PCR:

Beside the evaluation of the gel picture you are sending, the success of the PCR reaction serves as our quality control.  After the PCR and at various steps of the sequencing workflow further quality controls are performed.

NGSelect Amplicon Adaptor Ligation:

If the amount, concentration or quality of the starting material fails to meet the requirements for further processing, our customer care team will contact you to discuss how to proceed with your project. Whenever possible, Eurofins Genomics will recommend additional measures for optimising sample quality.

NGSelect Amplicon Adaptor Ligation:

Eurofins Genomics performs the 2nd PCR step with optimised PCR conditions and highest success rates. Still the success depends on various factors, including the amount of PCR product with universal adaptors. In case the 2nd PCR does not result in an amplicon and if we rate another trial as promising based on our experience, Eurofins Genomics will repeat the amplicon generation one-time. In case the repetition is not successful, Eurofins Genomics will stop the processing of the relevant samples and reimburse you for the sequencing part that was not performed. In the event that only a weak amplicon can be generated, Eurofins Genomics will include those amplicons in the amplicon pool for sequencing, too. Experience shows that those amplicons will result in very few reads only and interpretation of those reads should be done with special care.

Affordable high quality bioinformatics analysis is available for each package (see “Product Specifications” for more.

Yes. On request, the service can be performed under diagnostic conditions with ISO17025 certified processes.

For NGSelect Amplicon 2nd PCR the result files (FAST-Q files and analyzed data) can be downloaded from a secure FTP server (password will be provided), for NGSelect Amplicon Adaptor Ligation the results will be delivered via your Ecom account.

NGSelect is divided into four major categories, depending on starting material, DNA, RNA, amplicons or ready-to-load libraries. Each service offers a wide range of options that help create individualised and affordable NGS projects. Please see table 2 (below) for more information:

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

For a list of accepted starting material, including the relevant amounts, please refer to Table 1 in Product Specifications.

The quality and quantity of each incoming sample will be determined by appropriate methods (e.g. agarose gel analysis / Qubit® Fluorometer / NanoDrop / Agilent 2100 Bioanalyzer). Further quality controls are performed at various steps of the process.

If the amount, concentration and / or quality of the starting material do not meet the requirements for further processing, we will contact you to discuss how to proceed. If possible, Eurofins Genomics will recommend additional pre-processing steps in order to optimise the sample quality.

Affordable high quality bioinformatics analysis is available for each package including data filtering and QC.

Yes, upon request, the service can be carried out under diagnostic conditions with ISO17025 certified workflows.

All raw data (FAST-Q files) as well as the analysed data can be downloaded via your secure online account.

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

For a list of accepted starting material, including the relevant amounts, please refer to Table 1 in Product Specifications.

All ready-to-load libraries received are subject to a quality and quantity control to accurately determine the appropriate sequencing dilution and achieve optimal cluster densities.

If the amount, concentration or quality of the ready-to-sequence library does not meet the requirements for further sample processing, we will contact you to discuss how to go on with the project. Whenever possible, Eurofins Genomics will make recommendations for optimisation of sample quality.

Affordable and professional bioinformatics analysis is available for on request. For more information, see “Product specifications”.

Yes, on request, the sequencing service can be performed under diagnostic conditions that are ISO17025 accredited.

All raw data (FAST-Q files) as well as the analysed data can be downloaded via your secure online account.

The actual achieved sequencing output (read length, read quality and number of reads) is directly correlated with the quality of the sequencing library used. Eurofins Genomics cannot give any guarantees for sequencing data resulting from ready-to-load libraries prepared by the customer. If unsatisfactory sequencing results are due to technical problems with the sequencing kits or machines, the sequencing will be repeated at no additional cost to the customer.

Raw data sorting according to Illumina Index read is included. Used index type (single-indexed or dual-indexed) and corresponding index sequences have to be provided prior to project start.
Raw data sorting according to customer specific tags at read start can be offered at additional costs. We highly recommend to avoid the “in-line” barcoding strategy and use the Illumina index system instead (i.e. the barcodes are read in a separate read and do not interfere with cluster registration). It is important to ensure that the base composition of the indices is balanced to optimise the ability of the image analysis software to distinguish signals.

Illumina cluster detection algorithms are optimized around a balanced representation of A, T, G, and C nucleotides. Any divergence from equal base distribution will negatively influence the amount and quality of sequencing data produced. To increase the library nucleotide balance a spike-in of 20% PhiX will be used for samples with low diversity or unbalanced base composition (e.g., amplicons, bisulfite converted samples). The extent to which the negative impact of the unbalanced base composition will be reduced by spiking the PhiX control depends on individual sample and sequence characteristics. For more information please refer to the Illumina website.

NGSelect is divided into four major categories, depending on starting material, DNA, RNA, amplicons or ready-to-load libraries. Each service offers a wide range of options that help create individualised and affordable NGS projects. Please see table 2 (below) for more information:

For INVIEW Oncoexome Liquid Biopsy, we accept fresh or retained plasma samples as well as already isolated cell-free DNA. Please refer to our Liquid Biopsy sample preparation sheet (see "Files"). The ctDNA is extracted from the blood plasma fraction. 
For starting material such as DNA isolated from FFPE and tissue samples, please refer to our product Inview Human Exome.

In general we require either 4 mL of plasma or 15 ng already isolated cell-free DNA (up to 30 µl / concentration > 0,5 ng/µl).

Several pre-sequencing services are included in the Oncoexome product. These include ctDNA extraction from plasma and high- complexity library preparation with low duplicate rates. Exon targeting is accomplished with Agilent’s SureSelect platform, which offers superior exon enrichment and coverage uniformity.

The required coverage depends on the required sensitivity of variant detection. You may choose the sequencing coverage you need for your individual project study objective.
We typically recommend 100x coverage based on experience gained from analysing cell-free DNA from more than 40,000 samples to date.

The quality and quantity of each sample will be determined upon receiving the sample. Further quality controls are performed at various steps of the process.

All three tests are based on liquid biopsy. The three products serve as powerful non-invasive tools for real-time cancer profiling with the ability to capture the entire heterogeneity of the disease.

INVIEW Oncoexome Liquid Biopsy is the first commercially available service for whole exome sequencing (WES) of ctDNA. With this service, you can obtain a comprehensive profile of all mutations present in the protein-coding regions of the genome. This product is suitable for a preliminary look at the genome of a suspected cancer patient, in cases where limited information regarding clinically relevant mutations is available.

INVIEW Oncopanel Liquid Biopsy is a comprehensive NGS panel for profiling important cancer mutations. It uses single-molecule PCR to target 50 known cancer genes including tumour suppressors, mutation hotspots and drug-resistance markers. The panel sets a new industry standard in terms of sequence coverage and uniformity with a sensitivity of about 1%.

INVIEW Oncotarget facilitates ultra-sensitive detection of clinically relevant cancer-specific point mutations, insertions, deletions and gene fusions in circulating tumour DNA down to an allele frequency of 0.1%. This approach is suitable for treatment monitoring as it is sensitive enough to detect a relapse at a very early stage.

All deliverables (FAST-Q files and analyzed data) can be downloaded via your secure online account.

Blood samples have to be shipped to Eurofins Genomics within 24 hours after drawing blood. The sample must be stored in BCT Streck tubes. Samples may be stored and delivered at room temperature. Plasma samples have to be shipped on dry ice.

Eurofins Genomics accepts any number of samples in multiples of four.

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

For a list of accepted starting material, including the relevant amounts, please refer to Table 1 in Product Specifications.

Typically, a clearly defined Ensembl name for the annotated genomic reference sequence has to be provided before the project starts. Another option is to provide the genome sequence in Fasta format (along with the respective annotation in Gene transfer format (gtf)) or in GenBank format (including annotation).

The quality and quantity of each sample is determined by appropriate methods (e.g. agarose gel analysis / Qubit® Fluorometer / NanoDrop / Agilent 2100 Bioanalyzer). Further quality controls are performed at nearly all steps of the process.

If the amount, concentration or quality of the starting material does not meet the requirements for further sample processing, we will contact you to discuss how to go on with the project. If possible, Eurofins Genomics will make recommendation son how to optimise sample quality.

Affordable high quality bioinformatics analysis is available for each package including data filtering and QC.

Yes, upon request, the service can be carried out under diagnostic conditions with ISO17025 certified workflows.

All raw data (FAST-Q files) as well as the analysed data can be downloaded via your secure online account.

NGSelect is divided into four major categories, depending on starting material, DNA, RNA, amplicons or ready-to-load libraries. Each service offers a wide range of options that help create individualised and affordable NGS projects. Please see table 2 (below) for more information:

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

Depending on your choice of target regions, all bacterial, archeal and fungal species of which a sequence is published in the NCBI nucleotide database can be identified. There are currently for example 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.

• 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 therefore difficult and sometimes not possible.
• The more starting material we receive, the higher the probability to get excellent sequencing results.

• Samples with strongly degraded 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 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.

For bacterial profiling please choose at least on 16S target, for fungal profiling at least one ITS target. For taxonomic profiling or archea please selcet our archeal 16S target.

As each primer set fails to amplify certain species we recommend that you carefully check the literature available about the limitations of each primer set. It is recommended to use multiple primer combinations to capture the entire community (e.g. Ihrmark et al., 2012; Toju et al., 2012).

No. Our standard primer sets are not to be used for detection of cyanobacteria. If this is your aim, please inquire for an individual project.

We use the amplicon generation process as the entry quality control. Even though the PCR conditions are optimized the success of the amplicon generation depends on the amount of bacterial, archaeal or fungal DNA in the sample. In case we can not generate an amplicon or only a very week amplicon this is a strong indication that the content was too low. By default we will repeat the amplicon generation once with modified conditions. 

In case no amplicon can be generated, we will stop processing of those samples and reimburse you for the sequencing part that was not performed. In the event that only a weak amplicon can be generated, Eurofins Genomics will include those amplicons in the amplicon pool for sequencing, too. Experience shows that those amplicons will result in very few reads only and interpretation of those reads should be done with special care.

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.

The starting material is amplicons prepared in your lab that span the mutation site. The amplicon generation protocol to apply depends on the product type (adaptor ligation or 2nd PCR workflow), instructions can be found in the tab "How to prepare your Amplicons" on the respective product webpage. Amplicon length requirements can be found in the tab "Specifications".

For INVIEW CRISPR Check, the wildtype amplicon size needs to be adapted to the length of InDels you want to be able to analyse and must allow merging of read pairs. Usually mutations introduced via the NHEJ pathway are in a range of 1-20 bp, but in order to not miss the larger ones, the wildtype amplicons must be in a rather narrow size range. Length requirements for two use cases and all product types are available in our sample preparation guide.

Yes you can. In that case please provide the cleavage offset of your system (number of nucleotides upstream of PAM sequence where double strand break is expected).

1. Always include at least one unmodified (wildtype) sample as control
2. Include replicates if you aim for a very accurate prediction of the editing efficiency. Replicates increase accuracy of predicted editing efficiency, but are not strictly necessary.

Both product types are excellent solutions to analyse mutations introduced by CRISPR/Cas system (NHEJ pathway).

The products vary mainly in the sequencing library preparation (2-step protocol vs. adaptor ligation protocol) and thus in the way you are preparing amplicons for sequencing. 

A selection guide is available at the CRISPR website (tab Overview) to help you find the best solutions for your individual preferences.

• Comprehensive report with results and methods sections
• Raw FASTQ files
• Quality clipped and assembled FASTQ files
• Mapped reads in BAM format
• Observed wildtype and alternate allele sequences including their quantification 
• Raw data, preprocessing, mapping, and coverage metrics in CSV format
• Calculated Editing Efficiency per sample

For INVIEW CRISPR Check 2nd PCR the result files can be downloaded from a secure FTP server (password will be provided), for INVIEW CRISPR Check Adaptor Ligation the results will be delivered via your Ecom account.

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