Frequently Asked Questions About Our Products And Services

An overview of all our sample preparation guidelines can be found here >>

Please send your samples in barcode labelled 1.5 ml safe-lock tubes; or for > 48 samples in Eppendorf twin.tec PCR Plate 96, full-skirted, leaving position G12 & H12 empty.

Yes, we do perform a QC of the quantity of your sample.

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.

Please select your wished service (for example in the quick order menu on top of the page) and follow the instructions.

Depending on the selected product you might need to raise a quote first.

Once your quote is available you will be informed, and you can accept it in your account (please navigate to “Account” -> “Quotes”).

During the ordering process you will be asked to upload your samples via our Sample Submission Sheet. You can find the option to download the sheet directly next to the upload button (please always download the sheet here as it can differ between the products we offer). This sheet requires you to give us more information about your samples, as well as assigning Eurofins NGS Barcode Labels to your samples.

Our Eurofins NGS Barcode Labels can be ordered (free of charge) on our website.

Path: “Quick Order” -> “Next Generation Sequencing” -> “Additional Services” -> “NGS Barcodes & UPS labels”

After a few days you will receive your labels. Please stick them on your tubes based on the assignment you made on the Sample Submission Sheet. Please note that we can only accept samples arriving on our laboratory that are labelled with our Eurofins NGS Barcode Labels.

You can also find a list of your Eurofins NGS Barcode Labels at “Account” -> “NGS Barcodes & Coupons” -> “NGS Barcodes”.

Please note that Eurofins Genomics has different sites in Germany. Therefore, please make sure you are shipping to the correct site as instructed by your quote or sales representative.

If you require UPS labels you can order them on our website

Path: “Quick Order” -> “Next Generation Sequencing” -> “Additional Services” -> “NGS Barcodes & UPS labels”)

The UPS label will be send to you via Email within a day or two.

The default address (not S2 material, and no ONT Lite service) is:

Eurofins Genomics Europe Pharma and Diagnostics Products & Services Sanger/PCR GmbH
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Your order can be tracked in your Eurofins account.

Please navigate to your “Account” -> “Orders” -> “My Orders”.

Here you can see all your orders listed.

For more detailed information please klick on the Tracking Details icon (see below). It leads you to our Order Tracking page where you can find all your samples and their current status.

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.

Login to your Eurofins account with your e-mail address and password and click on “My Orders”, then on the icon next to your OrderID (see screenshot below).

You will find the files under section “DOWNLOAD DOCUMENTS & FILES”.

If you have received any compressed files, we recommend 7-ZIP (https://www.7-zip.org/) to uncompress them. Files will be deleted from our server 8 weeks after delivery.

Alternatively, you can access your data via our FTP server at ngs-ftp.eurofinsgenomics.eu using the username (the "ftp-" is part of the username) and password that you will receive in an email once your first data gets delivered. If you have forgotten your password, please enter ngs-ftp.eurofinsgenomics.eu to your browser and choose the "Forgot your password?" option.
 
Should you encounter any issues or have any queries, please do not hesitate to contact us.

We prioritize data security and make every effort to keep your data private and protected. Our data transfer methods are secure; however, if you prefer an alternative delivery method, we are happy to accommodate your request.

If you believe a processing error may have occurred, please contact us, and we will investigate on a case-by-case basis.

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

Depending on the product and if you ordered your service with our without additional bioinformatics analysis the data you receive may vary.

You will always receive FASTQ files and a HTML report. The additional data types can be viewed on the respective order page of the service you would like to order.

Here is an overview of all services we offer >>

This depends on the selected service and if you order with our without bioinformatics anlaysis.

You can view all bioinformatics details and the files you will receive on the content page of the respective service.

An overview of all NGS services we provide can be found here >>

• General FAQs for Next Generation Sequencing >>

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.

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).

INVIEW Human Exome Premium

Yes. 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).

INVIEW Human Exome Standard

No. The on target coverage cannot be guaranteed.

The target region corresponds to the bait coordinates of the Agilent SureSelect Human All Exon V8 Kit

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 (V8) 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.

• General FAQs for Next Generation Sequencing >>

For INVIEW Liquid Biopsy Oncoprofiling, 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 Oncoprofiling.

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, and CNVs.

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 Liquid Biopsy Oncoexome 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 Liquid Biopsy Oncoprofiling 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%.

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.

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.

• General FAQs for Next Generation Sequencing >>

For the INVIEW Oncoprofiling, 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 INVIEW Liquid Biopsy Oncoprofiling 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 728 cancer-relevant genomic regions, including protein-coding genes, select promoter regions, miRNAs, and extra-exonic variants. 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 INVIEW Oncoprofiling, 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, INVIEW Oncoprofiling'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 Oncoprofiling can accept conventional source material like fresh-frozen and FFPE tissues, whereas INVIEW Liquid Biopsy Oncoprofiling 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.

• General FAQs for Next Generation Sequencing >>

For INVIEW Metagenome 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.

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 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.

• General FAQs for Next Generation Sequencing >>

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.


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 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).

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.

• General FAQs for Next Generation Sequencing >>

NGSelect Amplicon on NovaSeq (150 - 270 bp):

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 on MiSeq (280 - 570 bp):

Beside the evaluation of the gel picture you are sending, the success of the PCR reaction serves as our quality control.  After the library prep 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.

Usually you would send per sample 1 amplicon generated with 1 primer pair. In some cases you might require however less reads per amplicon as given in the Project Specification. In such cases the individual amplicon samples you send may also consist of several amplicons. Please note, that in this case however we will not perform the clipping of primer sequences and deliver raw data only.

The adapter sequences are:

Forward: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’

Reverse: 5’-GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’

Yes, you can send in a single sample containing mixed amplicons for sequencing. Our Unique Sequence Analysis service is specifically designed to handle such samples. By using advanced sequencing and bioinformatics techniques, we can accurately separate and identify each unique sequence, ensuring you get comprehensive and reliable results from your mixed amplicon sample.

• General FAQs for Next Generation Sequencing >>

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.

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.

• General FAQs for Next Generation Sequencing >>

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.

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.

• General FAQs for Next Generation Sequencing >>

Common sample types include stool, saliva, skin swabs, environmental samples (soil, water), and more. Each sample type may require specific handling protocols. Please refer to our sample submission guide for more details. 

Microbiome profiling through next-generation sequencing (NGS) enables the detection of a wide range of species, including:

• Bacteria: All bacterial species with published sequences in the NCBI nucleotide database can be identified.
• Archaea: Archaeal species represented in the NCBI database can also be detected.
• Fungi: Fungal species can be identified through the analysis of Internal Transcribed Spacer (ITS) regions or 18S region, provided their sequences are available in the NCBI database.
• Cyanobacteria: Currently, identification of cyanobacteria is not possible due to the limitations of the existing primer setup. If cyanobacteria are a key focus for your study, we recommend exploring alternative primer sets or methodologies that are specifically designed for their amplification and detection.

Important considerations:

• Resolution Limits: Closely related species with nearly identical DNA sequences may not be distinguishable, and results will typically report only at the genus or family level.
• Sample Quality: The quality of the DNA and the specific regions targeted can impact the detection capabilities.
• Database Coverage: The identification of species relies on the completeness and accuracy of the reference sequences in the NCBI database.

Distinguishing Closely Related Species: NGS may struggle to differentiate closely related species with nearly identical DNA sequences, often resulting in identification only at the genus or family level.

DNA Quality and Degradation: The presence of degraded DNA, especially in highly processed samples or those with low pH, can lead to incomplete or inconclusive species identification.

Sensitivity Issues: While NGS is highly sensitive, this can also be a drawback. Low-abundance sequences or those with unexpected lengths may be detected but could be due to PCR or sequencing errors, requiring filtering out during data analysis.

False Positives/Negatives: Due to the method's sensitivity, there is a risk of false positives if sequences present at low levels are misidentified. Conversely, species present in very low abundance (typically below 0.5% of all assigned reads) may not be reported.

Dependence on Reference Databases: The accuracy of species identification relies heavily on the completeness and quality of reference sequences in databases like NCBI. Uncharacterized or poorly annotated species may be missed.

Primer Bias: Different primer sets may amplify certain species better than others, leading to an incomplete representation of the microbial community. It’s important to choose primers carefully based on their known limitations.

Yes, 16S primers can sometimes amplify plant organelles, specifically the 16S rRNA gene found in the chloroplasts of plants. Chloroplasts contain their own ribosomal RNA genes, which are similar to those found in bacteria due to their evolutionary origin.

However, the degree of amplification will depend on several factors, including:

• Primer Specificity: Some 16S primers are designed specifically for bacterial targets and may not efficiently amplify chloroplast DNA. Others may be more inclusive.
• Sample Composition: In samples with a complex mixture of organisms, such as environmental samples, the presence of plant DNA can lead to amplification of chloroplast sequences.
• Target Region: Different regions of the 16S gene may show varying levels of conservation between bacterial and chloroplast DNA, influencing the likelihood of successful amplification.

If you aim to specifically analyze bacterial communities and want to avoid amplifying plant organelles, it may be necessary to choose primers that are specifically designed to target bacterial 16S rRNA genes.

False Negatives:

• Degraded DNA: Samples with significantly degraded DNA may lead to no species identification, resulting in false negatives.
• Low Abundance: Species present at very low levels (typically below 0.5% of all assigned reads) may not be detected or reported, even if they are present in the sample.

False Positives:

• High Sensitivity: NGS detects all successfully amplified DNA amplicons. Sequences with low abundance or unexpected lengths may be incorrectly identified due to PCR or sequencing errors.
• Filtering Process: To mitigate false positives, stringent bioinformatics filters are applied during data analysis.

Negative Controls:

• To reduce false positives, a negative control (sterile water) is analyzed alongside samples.

When conducting 16S microbiome analyses, consider the following guidelines for selecting target regions and determining the number of targets:

Select Target Regions: For comprehensive microbial profiling, choose at least one target region from the 16S rRNA gene. Commonly analyzed regions include V1-V3, V3-V4, or V3-V5. The choice depends on your research objectives and the specific taxa of interest.

Consider Multiple Targets: To maximize coverage and capture the diversity of your microbial community, it’s beneficial to analyze multiple 16S target regions. This can help compensate for the limitations of individual primer sets, as some may fail to amplify specific species.

Literature Review: Review existing studies and literature related to your chosen target regions. This will provide insights into potential biases or limitations associated with different primer sets and can guide your selection.

Sample Type: Consider the nature of your samples (e.g., environmental, clinical) and their expected diversity. More complex samples may require a broader range of target regions to accurately reflect the community structure.

Experimental Design: If your analysis aims to compare different samples or conditions, ensure that the same target regions are used across all samples for consistency in data interpretation.

We utilize the amplicon generation process as our primary entry quality control. While our PCR conditions are optimized, the success of amplicon generation heavily depends on the amount of bacterial, archaeal, or fungal DNA present in the sample.

DNA Extraction Quality: If we extract the DNA, the quantity of extracted DNA is evaluated using spectrophotometry.

Initial Assessment: If we are unable to generate an amplicon or if the generated amplicon is very weak, it serves as a strong indication that the DNA content may be insufficient.

Negative Controls: Each batch includes negative controls (sterile water) to detect any potential contamination during sample preparation.

Sequencing Quality Assessment:

• Read Quality Monitoring: After sequencing, the quality of the reads is assessed using metrics like Q-scores. This helps identify any potential issues with sequencing accuracy.
• Filtering of Low-Quality Reads: Low-quality reads and adapter sequences are removed during the bioinformatics analysis to enhance data quality.

No Amplicon Generation:

If no amplicon can be generated, we will inform you and halt processing for those samples and reimburse you for the sequencing portion that was not completed.

Weak Amplicon Generation:

If only a weak amplicon is generated, we will inform you and Eurofins Genomics can include these amplicons in the sequencing pool on customer request. However, experience shows that weak amplicons typically result in very few reads. Interpretation of these reads should be approached with caution.

Genetic Variation: Different species have varying lengths of target regions within their genomes. For example, the 16S rRNA gene can vary significantly among bacterial species, leading to differences in amplicon lengths when amplified.

Intraspecies Diversity: Even within a single species, genetic variations can lead to differences in amplicon lengths due to polymorphisms or mutations.

Yes, especially regarding human samples. Ensure compliance with relevant ethical guidelines and obtain necessary approvals for research involving human subjects. 

• General FAQs for Next Generation Sequencing >>

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 (depending on the size of the amplicon), 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.

Usually you would send per sample 1 amplicon generated with 1 primer pair. In some cases you might require however less reads per amplicon as given in the Project Specification. In such cases the individual amplicon samples you send may also consist of several amplicons. Please note, that in this case however we will not perform the clipping of primer sequences and deliver raw data only.

• Whole Genome Sequencing (WGS) of small genome
• Full-length 16S sequencing
• Amplicon sequencing
• Whole plasmid sequencing

No this is not recommended. Optimal results can be achieved when the fragmentation is done directly before the library preparation. We recommend to send unfragmented HMW DNA, as large as possible.

This sample shipment instruction is for ONT products (not ONT Lite) with full flow-cells:

1. Whole Genome Sequencing Premium
2. Amplicon Sequencing (also complex amplicons)
3. 16S Full-Length Microbiome Sequencing

Instructions:

• Please use 1.5 ml safe-lock tubes for your templates and primers
• Do not tape or wrap tubes with parafilm. Safe-lock tubes offer perfect sealing and evaporation protection
• Label your template with our free NGS barcode labels.
  

If you send more than 92 samples then you can also sent the samples in plates. Please use full skirted PCR plates (e.g. Eppendorf or Twin).

For more information and for sample submission for extraction please read our Sample Submission Guide.

(Picture shows the procedure with Sanger Prepaid Barcodes but work the same with free NGS Barcodes)

If you like to order an ONT Lite product (Whole Plasmid Sequencing, Clonal Amplicon Sequencing, Bacterial genome sequencing up to 7 MB), please see here for detailed sample submission guidelines:  ONT Portfolio sample shimpent >>

Eurofins Genomics Europe Pharma and Diagnostics Products & Services Sanger/PCR GmbH
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Please note: this address is not correct for ONT Lite products!

For ONT Lite you can either use our dropboxes or send your samples to:

Eurofins Genomics
Sequencing Lab
Gottfried-Hagen-Straße 20
51105 Köln

Our ONT Lite portfolio is a highly automated high-throughput process. This enables fast turnaround times and great pricing.

Our complete ONT Lite portfolio can be seen here >>

Beside the ONT Lite portfolio we offer highly flexible and customisable services on Oxford Nanopores' GridION and PromethION machines. This ranges from premium whole genome sequencing (e.g. large bacterial genomes or eukaryotic genomes) to full-length 16S Microbiome profiling or non-clonal / complex amplicon sequencing projects.

To learn more about our capabilities, please click here >>

The panels are handled in batches of 88 or 384 samples.

Eurofins Genomics Europe Pharma and Diagnostics Products & Services Sanger/PCR GmbH
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Olink Explore is fully validated for human plasma and serum samples.

Other sample types, for example CSF, have been shown to be compatible with the technology

For most other sample types, Olink® Target 96 panels are recommended, due to the longterm experience of the OLINK team with the target panel. Example sample types: tissue and cell lysates, fine needle biopsies, exosomes, microdialysis fluid, bronchoalveolar lavage fluid, cell culture media, dried blood spots, synovial fluid, cerebrospinal fluid, plaque extract, urine, saliva and many more.

The Olink^® platforms are underpinned by a distinctive technology known as Proximity Extension Assay (PEA). This innovative dual-recognition, DNA-coupled methodology offers an unparalleled level of readout specificity. PEA empowers us to conduct high-throughput biomarker analysis with an extensive multiplex capability while maintaining uncompromising data quality standards.

On average, our turnaround time for bacterial genome sequencing, for genomes up to 7 Mb, is 3 to 5 business days.

Unfortunately not. This barcode is required for our internal lab processes and is essential in ensuring that we can process your samples as efficiently as possible. Thank you for your cooperation!

Yes of course.

If you don't know if there is a dropbox close by, use our dropbox location page to find out. Please click here >>

There are several reasons why your results might be different than what you expect.

To interpret your results you can have a look at our troubleshooting guide. Click here >>

For each run, we conduct multiple quality control (QC) measurements to assess the success of the library preparation and sequencing. If all the measurements indicate satisfactory results, we will not reprocess samples that have failed.

According to the specifications provided by Oxford Nanopore for the v14 library prep chemistry and the R10.4.1 flowcells currently employed in bacterial genome sequencing, the raw read accuracy exceeds 99%. The final assembly's accuracy fluctuates based on coverage and data quality. Generally, higher coverage, implying more reads for consensus construction, tends to enhance result accuracy.

Successful sequencing is determined by attaining at least one of the following outcomes:

1. A well-constructed genome assembly.
2. The specified quantity of raw data (equivalent to about 30x genome coverage of an average bacterial genome).

Deliverables:

• Data analysis report in HTML format
• Raw Sequencing data
• Assembled genome in FASTA format
• Circular genome plot
• Annotated genome sequence of all contigs in GENBANK format.
• Annotated genome features in GFF3 format
• Annotated feature table, including the nucleotide sequence of each feature
• Nucleotide sequences of all gene sequences in FASTA format
• Amino acid sequences of all annotated protein sequences in FASTA format

This service is designed for a clonal population (single strains) of bacteria. While you can submit mixtures of different bacterial strains for sequencing, it comes with a level of uncertainty in the assembly outcome, and therefore, it is done at your own risk.

If you are interested in mixed samples, we recommend using any of our other services:

• Microbiome profiling
• 16S Full-length sequencing
• INVIEW Resequencing of bacteria
• Whole genome sequencing on GridION (Illumina data polishing possible)

If your genomic DNA (gDNA) extract includes native plasmid DNA, it is likely that you will receive sequencing reads for those plasmids. Generally, DNA fragments with a size of <1 kb are excluded during sequencing. However, small plasmid-sized reads are not filtered out during assembly, so there is a probability that they will contribute to their own plasmid assemblies in addition to the gDNA assembly.

Ultimately, the types of DNA in your sample that contribute to an assembly will depend on the overall sample quality, coverage, and the relative abundance or degradation of each type of DNA.

Yes of course. Please check our full Oxford Nanopore portfolio here >> or send us your request >>.

A contig is the result of the assembly process, consisting of contiguous DNA sequences/ sequencing reads and not interrupted by unknown regions.

Even though it appears 100% perfect, there is still a likelihood of having single nucleotide polymorphisms (SNPs) in your sequence, especially in regions with homopolymers where Oxford Nanopore technology tends to have a higher error rate. To achieve a truly flawless assembly, we recommend using additional Illumina data to polish your results. Please feel free to contact us for further assistance.

The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.

So please handle these regions with special care.

For DNA sample preparation it is sufficient to use a high quality column based DNA preparation kit. No high molecular weight DNA is required.

• In order to offer our competitively priced product, we pool different samples on flow cell runs and re-use flow cells by washing them between runs. Both procedures are industry standard and performed according to the manufacturer’s specifications.  
• Barcoding and pooling samples on a single flow cell reduces cost and maximizes usage efficiency of the pores. However, nanopore sequencing has a higher error rate compared to “traditional” short-read next-generation sequencing, which can lead to misreadings in the barcode sequence and incorrect sample assignment/de-multiplexing at a rate of up to 0.05%. This issue is known as index hopping. 
• The flow cell washing procedure is very effective with only up to 0.1% of any previously loaded sample contaminating a subsequent run (known as carry-over contamination), nevertheless you might experience individual contaminating reads within your raw data.
• However, for both index hopping and carry-over it is important to note that a contamination of this level is unlikely to impact the final sequence assembly as several fine-tuned sequence cleaning and filtering steps are applied, provided your sample meets our quality and quantity standards and therefore yields sufficient numbers of high-quality amplicon reads. It is important to note, that the issue is most prominent in low coverage assemblies (see ‘What is the coverage?’).
• In the case that customer samples do not adhere to our sample specifications, the samples are sequenced on the customer’s own risk, and we cannot assume any liability for potential damages caused.
• If you’re concerned about contaminations and most importantly about reads from your samples appearing in other customer’s raw data, we offer the possibility to use our premium ONT services which include usage of a dedicated full flow cell for individual customers (learn more >>).

Unfortunately not. This barcode is required for our internal lab processes and is essential in ensuring that we can process your samples as efficiently as possible. Thank you for your cooperation!

Yes of course.

If you don't know if there is a dropbox close by, use our dropbox location page to find out. Please click here >>

There are several reasons why your results might be different than what you expect.

To interpret your results you can have a look at our troubleshooting guide. Click here >>

For each run, we conduct multiple quality control (QC) measurements to assess the success of the library preparation and sequencing. If all the measurements indicate satisfactory results, we will not reprocess samples that have failed.

The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.

So please handle these regions with special care.

The assembler may encounter challenges reconstructing the terminal ends of linear DNA, leading to the potential omission of approximately ~10 nucleotides from the 3' and/or 5' ends of your insert, depending on your sample's sequence.

If you observe this issue with your samples, you can address it by downloading the raw reads from your Dashboard and reconstructing the ends using your preferred method.

We do not provide a specific coverage guarantee. The number of raw reads generated can significantly vary based on sample quality. Typically, successful samples sent at the required concentration yield a range from high dozens to hundreds (or even thousands) of raw sequencing reads.

The average coverage of your sample is included in the result report HTML, where a coverage exceeding ~20x indicates a very accurate consensus.

Our amplicon service is designed for a population of molecules that is clonal.

What will happen if you send mixtures?

If your species are sufficiently distinct (e.g. vastly different in size or sequence), the pipeline will first attempt to make a .fasta consensus file from the highest abundance species. It will also attempt to make a consensus of other species with read counts that are >20% of that of the most abundant species. Ultimately, which species end up producing a consensus will vary depending on overall sample quality, coverage, and relative abundance/degradation of each species.

How can I sequence my amplicon mixture?

Eurofins Genomics offers a vast amplicon sequencing portfolio. You will definitely find the correct service for your needs: Amplicon portfolio>>

• In order to offer our competitively priced product, we pool different samples on flow cell runs and re-use flow cells by washing them between runs. Both procedures are industry standard and performed according to the manufacturer’s specifications.  
• Barcoding and pooling samples on a single flow cell reduces cost and maximizes usage efficiency of the pores. However, nanopore sequencing has a higher error rate compared to “traditional” short-read next-generation sequencing, which can lead to misreadings in the barcode sequence and incorrect sample assignment/de-multiplexing at a rate of up to 0.05%. This issue is known as index hopping. 
• The flow cell washing procedure is very effective with only up to 0.1% of any previously loaded sample contaminating a subsequent run (known as carry-over contamination), nevertheless you might experience individual contaminating reads within your raw data.
• However, for both index hopping and carry-over it is important to note that a contamination of this level is unlikely to impact the final sequence assembly as several fine-tuned sequence cleaning and filtering steps are applied, provided your sample meets our quality and quantity standards and therefore yields sufficient numbers of high-quality amplicon reads. It is important to note, that the issue is most prominent in low coverage assemblies (see ‘What is the coverage?’).
• In the case that customer samples do not adhere to our sample specifications, the samples are sequenced on the customer’s own risk, and we cannot assume any liability for potential damages caused.
• If you’re concerned about contaminations and most importantly about reads from your samples appearing in other customer’s raw data, we offer the possibility to use our premium ONT services which include usage of a dedicated full flow cell for individual customers (learn more >>).

Unfortunately not. The protocol used for the high-throughput Clonal Amplicon Sequencing service does not allow single-stranded DNA.

But of course we can sequence your ssDNA molecules with our custom workflow. More information can be found here >>

It really depends on the sample quality. We cannot guarantee the level of coverage at this time. A good sample submitted properly will typically yields hundreds or even thousands of sequencing reads. Consensus coverage depends on how many reads are full-length amplicons and how many, if any, degraded.

A coverage exceeding approximately 20x indicates a highly accurate consensus sequence.

A coverage below 20x can include inaccurate assembled amplicon sequences. The most common reasons for low quality coverage assemblies are listed in ‘Troubleshooting guide for failed samples’.

We recommend purifying your amplicons with commercially available kits based on DNA-binding beads or columns or enzymatic cleanup. DO NOT ship any primers with your samples or mixed into your samples.

• General FAQs for Next Generation Sequencing >>

Custom Long-Read Amplicon Sequencing dedicates an entire flow cell to your amplicons. As a result, this method is more time-consuming and costly compared to ONT Lite Clonal Amplicon Sequencing service, but it is highly suitable in the following cases:

• When you need additional services like 16S Microbiome Analysis, Unique Sequence Analysis, Variant Analysis, Sequence Cleaning, Host Removal, Pod5 raw data delivery 
• When your linear DNA sample is not clonal but instead contains a mix of different molecules. 
• When full-length, end-to-end reads are required, without any fragmentation.
• When full-length assemblies without missing terminal nucleotides are required.
• When a higher number of sequencing reads is needed beyond the typical output of the ONT Lite Clonal Amplicon Sequencing service. 
• When you need to obtain all raw reads generated from your sample. 

We recommend purifying your amplicons with commercially available kits based on DNA-binding beads or columns or enzymatic cleanup. DO NOT ship any primers with your samples or mixed into your samples.

Submit purified PCR products with a size range of 500 bp to 25 kb. The amplicons should ideally produce a single band on a gel for optimal results.

Yes, you can send in a single sample containing mixed amplicons for sequencing. Our Unique Sequence Analysis service is specifically designed to handle such samples. By using advanced sequencing and bioinformatics techniques, we can accurately separate and identify each unique sequence, ensuring you get comprehensive and reliable results from your mixed amplicon sample.

According to Oxford Nanopore's specifications for the chemistry and flow cells we currently use, the consensus accuracy is typically greater than 99.99%

The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.

So please handle these regions with special care.

• General FAQs for Next Generation Sequencing >>

We recommend purifying your DNA with commercially available kits based on DNA-binding beads or columns.

Coverage is calculated using the formula:  

(Read length) × (Total number of reads) ÷ (Genome size). 

For example, sequencing a genome of 10 Mb with 20 million reads at 2 × 150 bp read length results in a coverage of 60×. "

Depending on your needs we recommend the following coverage: 

• Germline/frequent variant analysis: 20-50x 
• Somatic/rare variants: 100-1000x 
• De novo assembly: 100-1000x