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.

The entry QC performed depends on the type of product. For example for RNA-Seq we do not only check the quantity, but also the quality of the 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.

Samples that fail to meet the Incoming Quality Control (IQC) criteria (as defined on our website) may be canceled upon receipt of the IQC report, canceled with replacement samples sent, or processed further at the client’s own risk (in which case the read guarantee is no longer applicable).

If samples pass the Incoming Quality Control but subsequently fail the Library Quality Control, Eurofins will cover the costs of reprocessing the sample. If no sample material is available, we kindly request that you send us new starting material, if possible.

Depending on the type of virus or infectious material in your samples, we may be able to accept them. Eurofins can work with biosafety level L1 and L2 specimens. We do not accept samples with higher biosafety level than S2. GMOs are only accepted with S1 level. For RNA isolation service, please refer to our “Sample preparation and shipping guide for Extraction”.  

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.

Eurofins NGS Team is composed of Ph.D. scientists who can help you optimize your project design and provide consultation. Contact the team directly; our contact information is at the bottom of the page.  

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 >>

The total base output of long read sequencing is highly dependent on input DNA fragment length and quality.

To estimate the coverage for long read sequence the formula is used:  

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

For example, sequencing a genome of 50 Mb with 1 million reads and an average read length of 5 kb (total base output of 1 Gb) will result in an average coverage of 100x.

• 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  

• General FAQs for Next Generation Sequencing >>

We recommend: New England Biolabs Monarch® Spin gDNA Extraction Kit, QIAGEN Genomic-tip-500/G, QIAGEN MagAttract HMW DNA Kit, QIAGEN Puregene Yeast/Bacteria Kit. In general, all extraction methods that allow the generation of HMW gDNA fulfilling the above requirements can be used. To ensure to extract HMW DNA we recommend

• Wash bacterial cell pellets before DNA extraction with PBS to remove potential inhibitors
• Add 1ml of PBS and resuspend cells by pipetting
• Pellet the cells by centrifugation and discard the supernatant
• Avoid vortexing and fast or unnecessary pipetting; use wide-bore tips only
• Elute in nuclease-free elution buffer, not water
• Avoid over-drying of gDNA
• Do not expose DNA to high temperatures (>37°C) for >1 hour
• Use buffer with appropriate pH 7.5- 8.5
• Avoid intercalating fluorescent dyes, or UV radiation
• Avoid freeze-thaw cycles; store gDNA at 4°C for 1-2 months

Short DNA fragments are typically not ideal for long-read sequencing, as they can result in poor assembly quality. We recommend that at least 50% of the DNA is above 15 kb in length. To improve sequencing outcomes, we recommend removing shorter DNA fragments from your sample before submission. One effective protocol involves using 4X diluted SPRISelect beads (35% volume by volume) to eliminate fragments smaller than 3-4 kb.

Please note that the DNA quantity will decrease following this process, so it’s essential to quantify the remaining DNA to ensure sufficient material is available for sequencing.

• Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
• Dilute SPRISelect beads with Elution Buffer to 35% (volume by volume).
• Resuspend the diluted SPRISelect beads by vortexing.
• Add 4x volume of the resuspended diluted SPRISelect Beads to each gDNA and mix by flicking the tube.
• Incubate for 5 minutes at room temperature.
• Prepare sufficient fresh 80% EtOH in nuclease-free water for all of your samples.
• Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
• Keep the tube on the magnet and wash the beads with freshly prepared 80% EtOH without disturbing the pellet. The EtOH volume must be enough to cover the beads entirely. Remove the EtOH using a pipette and discard.
• Repeat the previous step.
• Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual EtOH. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
• Remove the tubes from the magnetic rack and resuspend the pellet in e.g. 50 µL nuclease-free 1xTE or EB. Spin down and incubate for 10 minutes at 37°C with gentle agitation (700rpm) on a shaker.
• Pellet the beads on a magnet until the eluate is clear and colourless.
• Remove and retain the eluate into a clean 1.5 ml Eppendorf Safe Lock Tubes™

Alternatively, we recommend the DNA clean-up and size selection for long-read sequencing from Jones et al. 2021 (Jones A, Torkel C, Stanley D, Nasim J, Borevitz J, et al. (2021) High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. PLOS ONE 16(7): e0253830. https://doi.org/10.1371/journal.pone.0253830; https://www.protocols.io/view/dna-clean-up-and-size-selection-for-long-read-sequ-261ge6ymol47/v4)

Purity:

• Phenol, chloroform and other related reagents will inhibit the library preparation and must be completely removed. Although spectrophotometric measurements may not detect all types of contamination, we highly recommend performing them to assess sample purity. Ideally, the 260/280 ratio should be above 1.8, and the 260/230 ratio should fall between 2.0 and 2.2. If the sample is contaminated and does not meet this metric, either re-extract the sample or clean up the sample to remove the contaminants using a Qiagen cleanup kit or AMPure XP beads.Must not contain RNA; we strongly recommend RNase treatment during extraction
• Must not contain denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
• Must not contain residual contaminants from the organism/tissue (heme, humic acid, polyphenols, etc.)
• Must not contain insoluble material or be colored or cloudy

Quantitative assessment 

Preferred measurement method: fluorescence-based methods like Qubit® (Invitrogen, Life Technologies), Quant-iT™ (Invitrogen) or Quantifluor (Promega). 

We strongly discourage using Nanodrop for gDNA quantification.

HMW gDNA often requires extra homogenization effort (longer incubation time, increased incubation temperature, very extensive gentle mixing, etc.) to obtain accurate quantification. If separate DNA quantifications from the top and bottom of the sample are within 15% of each other, this is usually a good indication of adequate homogeneity.

If you have less than the requested amount of gDNA, we strongly recommend that you perform additional extractions to increase yield.

Qualitative assessment 

Preferred measurement method: capillary electrophoresis-based methods like Fragment Analyzer or Bioanalyzer. High molecular weight DNA is greater than 15 kb in size and shows minimal smearing. Contamination, damage and degradation are revealed through a low molecular weight smear and should be removed using alternative cleanup strategies as described above. 

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

Depending on your needs we recommend the following coverage: 

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

Sequencing output is dependent on a variety of factors e.g. organism, extraction method, DNA quality, size, handling & storage. Therefore, successful EntryQC cannot guarantee high sequencing yield as not all inhibitors can be detected.

Plants, especially, can contain high levels of secondary metabolites that can interfere with the sequencing process. These compounds can be challenging to detect and remove during preprocessing, leading to potential issues in sequencing yield and data quality.

Based on our experience, you can generally expect around 50-80 Gb of data per PromethION flow cell and approximately 6-7 Gb per GridION flow cell. These yields are dependent on factors such as DNA quality and fragment length.

According to Oxford Nanopore's specifications for the chemistry and flow cells we currently use, raw read accuracy is >Q20 (>99%) and the consensus accuracy is typically greater than 99.99% respectively.

See https://nanoporetech.com/platform/accuracy

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.

No, we do not have any guarantees for assembly quality. However, using high-quality input DNA with small and not overly complicated genomes, a single contig is usually generated.

Available on request.

• 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).

We recommend starting with total RNA (DNA-free). For detailed sample submission requirements please visit our Sample Submission Guidelines.

To successfully extract RNA for RNA sequencing please follow these guidelines:

• Use Quality Reagents: Choose high-quality reagents and kits specifically designed for RNA extraction to ensure maximum yield and purity.
• Tissue or Cell Handling:
  • If using tissue, immediately snap-freeze or store it in RNA preservation solutions (like RNAlater) to prevent RNA degradation.
  • For cell cultures, process samples promptly and keep them on ice until extraction.
• Homogenization:
  • Homogenize the samples thoroughly to ensure complete lysis of cells and release of RNA.
• Follow Protocols Carefully: Adhere strictly to the manufacturer's protocol of the RNA extraction kit. Pay close attention to:
  • Sample input amount
  • Lysis buffer volume
  • Centrifugation times and speeds
• Avoid RNases:
  • Use RNase-free tubes, pipette tips, and reagents. Wear gloves and use sterile equipment to minimize contamination.
• Check RNA Quality and Quantity:
  • Use a spectrophotometer (e.g., NanoDrop) and a bioanalyzer / Fragment Analyzer to assess RNA purity (A260/A280 ratio) and integrity (RNA Integrity Number, RIN).
• Storage:
  • Store extracted RNA at -80°C for long-term preservation. For short-term storage, RNA can be kept at -20°C.
• Prepare for Library Construction:
  • Ensure that the RNA is free of contaminants (like DNA and proteins) and within the required concentration range for downstream applications.

The integrity of the sample material has a critical impact on the analysis results. Degraded RNA can lead to incomplete or inaccurate sequencing data, as fragmented RNA may not be efficiently captured or aligned. This affects gene-body coverage, resulting in uneven sequencing depth across the gene. Degraded RNA typically shows reduced coverage, particularly in the 3' and 5' regions of genes, leading to biased or incomplete gene expression profiles. High-quality, intact RNA is essential for consistent gene-body coverage and reliable, reproducible RNA sequencing results.

In particular, for poly-A enrichment, degraded RNA can introduce a strong bias towards the 3' end of the transcript. For these samples, we recommend rRNA depletion to improve data quality and reduce this bias.

You will be informed if your sample material does not meet our IQC criteria and will be provided with information about available options. In certain cases, processing at your own risk may be offered. In this case, guarantees for the successful creation of the library and sequencing will be void. If the library fails the quality control prior to sequencing, our Customer Care team will also contact you.

When processing sample material beyond the specs, several issues may arise and should be carefully considered:

• Decreased RNA Quality: Sample material that deviates from optimal conditions (e.g., improper storage, extended handling, or delays) may lead to RNA degradation, resulting in poor quality data.
• Lower Input Quantity: Reduced input RNA quantity can lead to insufficient material for accurate analysis, potentially causing biased or incomplete results due to low coverage or poor detection of lowly expressed genes. These samples may also fail to produce sufficient amounts of libraries for subsequent sequencing.
• Bias in Data: Alterations in sample handling or processing can introduce biases in gene expression profiles, such as uneven gene-body coverage, particularly in the 3' or 5' ends.
• Reduced Sensitivity: Handling samples outside of recommended conditions can affect the detection of lowly expressed genes, making the analysis less sensitive.

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.

Since ribosomal RNA (rRNA) constitutes the majority of a total RNA sample, we recommend removing it to enrich the sample for other RNA types. This can be achieved through either a poly-A selection or rRNA depletion method, both of which help to focus the analysis on more informative RNA populations

RNA sequencing (RNA-seq) is a powerful tool for transcriptome analysis, and choosing between polyA enrichment and rRNA depletion depends on the specific goals of the experiment and the types of RNA you wish to study. Here’s a breakdown of when each method makes sense:

1. PolyA Enrichment

PolyA enrichment selectively isolates mRNA by binding to the polyA tails found in the 3’ end of most eukaryotic mRNAs. This is useful when you are primarily interested in coding RNA (mRNA), and it simplifies the analysis by reducing the complexity of the sample.

When to use PolyA Enrichment?

• Focus on mRNA: If your goal is to analyze protein-coding genes, polyA enrichment is ideal because it captures most of the mRNA that has a polyA tail (which is the majority of the transcriptome in eukaryotic cells).
• Eukaryotic Samples: Most eukaryotic mRNAs have polyA tails, making polyA enrichment particularly useful for these species.
• Reducing rRNA Interference: In RNA samples with high rRNA content, polyA enrichment helps reduce rRNA contamination by selectively enriching for mRNA, although it won’t remove rRNA entirely.
• mRNA Profiling: If you are performing transcriptomics to understand gene expression levels or alternative splicing in coding regions, polyA enrichment is effective.
• Limitations:
  • Non-Polyadenylated RNAs: It won’t capture non-polyadenylated RNA, such as long non-coding RNAs (lncRNAs), miRNAs, rRNAs, and some small RNAs.

Considerations for Low-Quality RNA (PolyA Enrichment):

• Reduced mRNA Capture: If the mRNA is fragmented or degraded, polyA enrichment capture primarily the 3´end of mRNA, and there could be a loss of important data. This is often the case for RNA extracted from FFPE slides.
• Moderate Degradation: PolyA enrichment can still be useful when the RNA degradation is moderate (i.e., not severely fragmented), as long as there is a sufficient amount of intact polyadenylated RNA.

 2. rRNA Depletion

rRNA depletion removes the vast majority of ribosomal RNA (rRNA), which makes up 80-90% of the total RNA in most eukaryotic cells. By depleting rRNA, you can analyze other types of RNA that may be less abundant, such as mRNA (in cases where polyA enrichment is not preferred), lncRNAs, small RNAs, and other non-coding RNAs.

When to use rRNA Depletion?

• Comprehensive Transcriptome Analysis: If your goal is to study all types of RNA, including non-coding RNAs (like lncRNAs and miRNAs), small RNAs, or even non-polyadenylated mRNAs, rRNA depletion is a better option because it preserves these RNA types.
• Prokaryotic Samples: In prokaryotes (which have rRNA as the most abundant RNA), rRNA depletion is commonly used to improve the quality of the RNA-seq data, as prokaryotic mRNAs do not have polyA tails.
• Limitations:
  • Partial rRNA Removal: In some cases, rRNA depletion methods may not remove all rRNA species, leaving some residual.

Considerations for Low-Quality RNA (rRNA Depletion)

• Severe RNA Degradation: rRNA depletion is better suited for low-quality or degraded RNA, e.g. RNA extracted from FFPE slides, because it doesn’t rely on the integrity of polyA tails. Even with fragmented RNA, rRNA depletion can effectively reduce rRNA contamination.

Summary:

• PolyA Enrichment is optimal when you are focusing on mRNA and the RNA quality is high to moderate. It is less effective when RNA is degraded or when you need to analyze non-polyadenylated RNAs.
• rRNA Depletion works well for all non rRNA types and prokaryotic mRNA, especially if you want to capture a broader spectrum of RNA, including non-polyadenylated RNAs like lncRNAs. This method is also ideal when working with severely degraded RNA.

We offer rRNA depletion using specialized kits for a variety of organisms, including human, mouse, rat, bacteria, and plants. These kits are tailored to efficiently remove ribosomal RNA, allowing for a more focused analysis of the transcriptome in these species. 

Our rRNA depletion service is compatible with a wide range of mammalian species, though the depletion efficiency may vary depending on the species. Below is a summary of rRNA depletion efficiencies for several species based on estimates:

• Chicken: 82%, Cow: 98%, Cynomolgus monkey (Macaca fascicularis): 88%, Dog: 90%, Hamster: 95%, Horse: 98%, Pig: 80%, Rabbit: 81%, Sheep: 95%

Our rRNA depletion service is compatible with a wide range of plant species, though the depletion efficiency may vary depending on the species. Below is a summary of rRNA depletion efficiencies for several species based on estimates:

Arabidopsis: 99%, Barley: 92%, Corn: 90%, Cotton: 99%, Flaxseed: 87%, Maple: 89%, Oat: 99%, Potato: 93%, Rice: 91%, Rye: 99%, Sorghum: 79%, Soybean: 93%, Wheat: 94%

For RNA analysis from blood samples, we recommend both rRNA depletion and globin mRNA depletion. Since globin mRNA makes up a significant portion of the RNA in blood, depleting it alongside rRNA helps to enrich the sample for other less abundant but biologically relevant RNA species.  

For Ultra-Low Input RNA-Seq, we use poly-A selection to enrich for mRNA, as it is highly effective in capturing the transcriptome from small RNA quantities. Please note that rRNA depletion is not available for this service due to the limited amount of input RNA.

As a consequence, Ultra-Low Input RNA-Seq is only available for eukaryotic RNA.

The required number of reads depends on factors such as genome size, the number of known genes, and transcripts. As a general guideline, we recommend 5-10 million read pairs per sample for small genomes (e.g., bacteria), and 20-30 million reads per sample for larger genomes (e.g., human, mouse). For medium-sized genomes (e.g., yeast), the required number of reads can vary based on the project, but we typically suggest between 15-20 million reads per sample. For de novo transcriptome assembly projects, we recommend around 100 million reads per sample. 

We provide raw data as FASTQ files for all projects. We also have advanced bioinformatics capabilities to provide optional data analysis services. The bioinformatics services available for RNA-Seq projects are as follows:

• Standard Analysis:
  • Quality assessment of sequencing data
  • Alignment of reads to a reference genome
  • Assembly and quantification of transcripts
  • Normalization of gene counts
  • Identification of differentially expressed genes
  • Principal component analysis (PCA)
  • Significance testing
  • Generation of detailed reports and visualizations
• Optional Analysis:
  • Alternative splicing (AS) analysis
  • Gene fusion analysis
  • Variant detection and its effect on protein alteration
• Advanced Analysis:
  • Gene Set Enrichment Analysis (GSEA)

Each service includes specific deliverables and has associated costs and turnaround times.

When total RNA is sequenced, you can expect to detect a variety of RNA types, including:

• mRNA: Provides gene expression information.
• rRNA: Dominates most RNA samples, unless depleted.
• tRNA and non-coding RNAs: Smaller amounts, but play important regulatory roles.

If no specific enrichment is done, rRNA will likely dominate the data.

During the RNA-Seq – Ultra Low library preparation process, primers and adapter sequences are added to the RNA during cDNA synthesis and amplification. These sequences are necessary for the sequencing process, but they are not part of the actual biological transcript. If left in the data, they can introduce noise and reduce the quality of the sequencing results. To ensure high-quality data, Eurofins will trim these sequences prior to delivery, which may result in slightly shortened sequencing reads. 

• 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 >>

Submit at least 25 µL purified PCR products sized 150-270 bp, with a concentration > 4ng/µL with a purity (A260/280) of 1.8-2.0. Amplicons must not contain TruSeq adapter sequence.

Submit at least 25 µL purified PCR products sized 280-570 bp, with a concentration > 1ng/µL with a purity (A260/280) of 1.8-2.0. Amplicons have to contain a TruSeq adapter sequence.

Samples can be in RNase-, DNase- and protease-free water, EB, or low TE buffer.

Please also refer to “2. Ordering & Sample Shipment” to read more about organizational topics on how your samples need to be prepared before being shipped to our sites

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 150 – 270 bp (NovaSeq) or 280 – 570 bp (MiSeq). The amplicons should ideally produce a single band on a gel for optimal results.

No, only amplicons within our size ranges 150 – 270 bp (NovaSeq) or 280 – 570 bp (MiSeq)) are accepted. For larger products, please consider our ONT amplicon sequencing services. If you are unsure about product selection, please reach out to your sales representative.  

For 150-270 bp amplicons sequenced on our NovaSeq, please use our Inline Tag Design Tool : NGS Adapter Ligation Oligos (eurofinsgenomics.eu). When ordering the service, select the optional service “Tag sorting – Eurofins Genomics Design”. You will receive sorted sequences (FASTQ).

• 192 individual inline tags are available, each consisting of 10 nucleotides added to the forward primer.
• There is a minimum edit distance of 4 to prevent misassignments due to sequencing errors.
• The tags are optimized for both low and high sample pools.
• All NGSgrade Oligos undergo specific processes to ensure cross-contamination-free and high-purity performance.
• For multiplexing 24 samples with the same target sequence, 24 forward primers (each with a unique inline tag) and one reverse primer (without tags) are required.

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 >>

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.

• Raw Nanopore amplicon data (FASTQ) 
• Raw Nanopore sequencing data (POD5) available on request (additional costs) 
• Customizable data analysis packages are available in our ordering form
  • Variant analysis
    • Variant (SNPs & InDels) Calls (TSV)
    • Analysis Report (HTML)
  • 16S Microbiome Analysis
    • OTU Quantification Tables (TSV)
    • Analysis Report (HTML)
  • Unique Sequence Analysis
    • Unique Sequences (FASTA)
    • Clustering quantification table (TSV)
    • Analysis Report (HTML)
  • Consensus Sequence Analysis
    • Consensus Sequence (FASTA)
    • Variant Calls (TSV)
    • Analysis Report (HTML)

Various analyses can be selected during ordering, including 16S microbiome, consensus sequence analysis, variant detection and unique sequence identification, are available depending on your project's specifics. If you require a special analysis, please reach out to your sales representative. 

• 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 >>

All ready-to-load library pools are subject to a quality and quantity control to accurately measure the insert size and DNA concentration to determine 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 free of charge. Used index type (single-indexed or (unique) dual-indexed) and corresponding index sequences have to be provided prior to project start in the Sample Submission Form. 

Yes, we offer de-multiplexing of in-line barcodes in sequencing reads (not index reads). 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.

Please contact us for further details. Additional charges apply

You can submit any library pool compatible with Illumina® sequencing, including those prepared from genomic DNA for whole genome sequencing, RNA for transcriptome analysis, or amplicon libraries. Please note, pooling of different libraries into a final ready-to-load pool must be performed by our customers, this service is not offered by Eurofins Genomics.

Illumina-compatible libraries must include four key components in their adapter sequences—located at the ends of each fragment—for successful paired-end sequencing:

• Flow cell binding sites (P5 and P7): These enable the DNA fragments to attach and cluster on the flow cell.
• Sequencing primer binding sites (Read 1 and Read 2): These sites initiate sequencing from both ends of the DNA fragment.
• Index sequences (i5 and i7): Also known as barcodes, these allow for sample multiplexing. Libraries may be single-indexed (one index) or dual-indexed (two indexes). The usual length of barcodes for NovaSeq is in the range of 8 to 12 bp, while for MiSeq we recommend to use at maximum 10 bp index sequences.
• Index primer binding site(s): These are essential for reading the index sequences during sequencing.

Single-indexed library

Dual-indexed library

The structure of the adapter sequences varies depending on whether the library uses single or dual indexing. Missing, misconfigured, or heavily modified components can cause sequencing to fail. If you're unsure about your library’s configuration, feel free to contact us for guidance.

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 (MiSeq) 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.

Yes, we can do optional data analysis. Please indicate this on the quote request form.

• 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 >>

• 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.