w10.0.23 | c9.0.87.103
PROD | u7.5.14
Header Image service-corner
 

 

print content
increase font
decrease font

Frequently Asked Questions About Our Products And Services

The right answers to frequently asked questions

Find the answers to all our products and services by clicking the links below.

Next Generation Sequencing

Questions on Inview De Novo Genome

Where do I get my results?

All raw data as well as the analysed and assembled data can be downloaded via your secure myGATC online account. Example data of de novo assembly of E. coli DH1 can be viewed in “Files”.

What coverage should I use?

Internal tests showed that a coverage of about 25-30x with corrected PacBio RS II reads is sufficient to produce high-quality genome assemblies. This corresponds to raw data coverage of approx. 100x that should be achieved. Assembly results highly depend on the composition of the analysed genome and may vary between different organisms. 

Which starting material should I send and how much of it?

For sequencing on the PacBio RS II it is crucial that high-quality DNA is used as starting material. The use of too little, degraded, contaminated or otherwise damaged starting material can result in low yield or failure of the sample preparation and impair quality and amount of sequencing results. For optimal results we require at least 10 µg double-stranded, purified, high molecular and RNA-free DNA (concentration approx. 80 ng/µl; OD 260/280 ≥ 1.8; OD 260/230 ≥ 2.0.).

Do you guarantee a certain output?

Sequence data are assembled de novo under consideration of all read information, using optimized programs and parameters. The number of contiguous sequences (contigs) that can be unambiguously assembled depends on the complexity (frequency, length and distribution of highly repetitive and duplicate regions) of the sequenced genome and also the quality of the provided starting material. Therefore we cannot guarantee a minimum number of contigs.

What kind of quality controls do you perform?

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.

Do you have any examples of successful joint ventures?

Inview De Novo Genome 2.0 was applied within the KLEBSICURE Consortium, a cooperation project with GATC Biotech AG, the Max Planck Institute for Infectious Biology (Berlin, Germany) and the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy (Wroclaw, Poland) as consortium members and Arsanis Biosciences GmbH (Vienna, Austria) as consortium leader. 
The unique combination of de novo sequencing and detection of base modifications was used to study the virulence of the pathogen, to guide the generation of monoclonal antibody therapeutics,and to establish a test method for clinical diagnostics.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Epigenome BS

Where do I obtain my results?

All raw data as well as the analysed and assembled data can be downloaded via your secure myGATC online account. A demo data report for a typical BS-Seq project can be found under the section “Files”

What coverage should I use?

Sequencing results can vary depending on the composition of the analysed genome and the organism being sequenced. Typically, 30x average genome coverage is recommended.  

What starting material should I send and how much of it?

For sequencing on the Illumina platform, we require 2 µg high-quality DNA that has not been amplified or bisulfite treated as starting material.

Do you guarantee a certain output?

We guarantee 20 million read pairs (+/- 10%) with 5 Gb raw data output (+/- 10%) per package.

What kind of quality controls do you perform?

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

Do you have any examples of successful joint ventures?

GATC Biotech is a partner in the EpiFemCare project. The project is partially funded by the EU’s Seventh Framework Programme (FP7) with the goal of using epigenetic-based methods to improve diagnosis and treatment measures for women with breast and ovarian cancer.
GATC Biotech manages sample processing, i.e. the DNA isolation from serum/tissues, bisulfite treatment and library preparation. Cutting-edge technologies in epigenetics, next generation sequencing and data management are applied to develop blood tests, with the aim of reliably detecting cancer and improving personalised treatment.

Where do I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Human Exome Advance

What is the data handling process, including QIAGEN’s Ingenuity Variant Analysis?

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

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

How long is my data accessible at my QIAGEN account?

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

Where do I get my results, if I only choose the optional BioIT from GATC Biotech?

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

What coverage should I use?

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).

How do you handle PCR duplicates?

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 GATC Biotech.
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.

Which starting material should I send? In what quantities?

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

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

What do you mean by overlapping reads?

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, GATC is continuously optimising its protocols to minimise the number of overlapping bases.

What is the quality of the data?

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

Do you guarantee a specific on-target output?

For samples that have successfully passed the initial quality check at GATC Biotech, 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).

Which genes are enriched?

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

Are all genes covered at 30x?

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. GATC Biotech applies the most recent Agilent Human All Exon kit design (V6) with improved design algorithms to better capture difficult regions and provide superior coverage uniformity.

What kind of quality controls do you perform?

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.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Human Exome Explore

What kind of samples can be processed with Inview Human Exome Explore?

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

How much starting material should I send?

Data output only applies for freshly isolated, unamplified and pure human DNA samples (minimum 100 ng). Please note that contamination by DNA of other species (> 3 %; especially from closely related organisms) reduces the total amount of human DNA in the provided sample and it might interfere with the enrichment of the exonic region.

Which genes are enriched?

The target region corresponds to the bait coordinates of the Agilent SureSelect Human All Exon V6 Kit: 60.7 Mbp of exonic bases covering >20,000 genes in the human genome.

How do you handle PCR duplicates?

PCR duplicate rates are directly correlated to the quality and amount of the provided starting material. Hence, the PCR duplicate rate is dependent on the sample and cannot be influenced by GATC Biotech.

Are all genes covered with 10 Mio read pairs?

Due to varying efficiencies of the enrichment baits (e.g., GC / AT content) targeted regions are covered differently and the range and coverage varies over the target region. GATC Biotech applies the latest Agilent Human All Exon kit design (V6) with improved design algorithms to better capture hard-to-capture regions providing superior coverage uniformity.

Based on our experience with the sequencing of human samples we typically receive 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 remaining duplicates are excluded from further analysis to prevent bias.

What do you mean by overlapping reads?

The fragmentation of genomic DNA according to the Agilent SureSelectXT2 protocol and downstream processes produces a Gaussian distribution of DNA fragment sizes. A small percentage of the resulting fragments have an insert size below 300 bp. Sequencing of these small library fragments with 150 bp paired end reads will generate partially overlapping reads which cover the same bases and hence create an artificial doubling of coverage at those positions. To assure accurate analyses, these bases are excluded from further downstream analyses including single nucleotide variant (SNV) and insertion and deletion (InDel) detection. GATC Biotech is continuously working on optimising protocols in order to minimise the number of overlapping reads.

What is the quality of the data?

For human samples sequenced with 150 bp paired-end mode, GATC Biotech typically achieves over 80 % of base calls with a quality value higher than Q30 ( >99.9 % accurate).

Where do I get my results and in which form are they delivered?

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

How does data processing with QIAGEN’s Ingenuity Variant Analysis work?

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

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

How long is my data accessible at my QIAGEN account?

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

How should I choose between Inview Exome Explore and Inview Exome Advance?

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

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

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

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on GATCLiquid Oncopanel All-In-One

What kind of samples can be processed with GATCLiquid Oncopanel All-In-One?

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

How much starting material is required?

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

How is the target enrichment performed?

The genes of interests are enriched via proprietary GATC 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 GATC 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.

What kind of mutations can be detected with GATCLiquid Oncopanel All-In-One?

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

What is the sensitivity of the product?

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

What kind of quality controls do you perform?

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.

How should I choose between GATCLiquid Oncoexome, Oncopanel All-In-One and Oncotarget?

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

GATCLiquid 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.

GATCLiquid Oncopanel All-In-One 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 GATC 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%.

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

Can I use GATCLiquid Oncopanel All-In-One for diagnostic purposes?

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

What are the limitations of the CNV analysis?

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.

Will all copy number variations in my sample be detected?

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 GATC Biotech’s highly sensitive methods.

Where do I get my results?

All deliverables can be downloaded via your secure myGATC online account.

How soon after the blood draw should I ship the sample?

We recommend that the blood sample be shipped to GATC Biotech 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.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Oncopanel All-In-One

What kind of samples can be processed with Inview Oncopanel All-In-One?

For Inview Oncopanel, we accept fresh-frozen and FFPE tissue and blood samples.

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

How is the target enrichment performed?

The genes of interests are enriched via a proprietary GATC protocols using Agilent SureSelect chemistry. The hybridisation-based strategy eliminates PCR-bias and reduces the number of PCR duplications. The proven target enrichment method has been optimised by GATC Biotech 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.

What kind of mutations can be detected with Inview Oncopanel All-In-One?

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

What is the sensitivity of the product?

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

What kind of quality controls do you perform?

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.

What is the difference between Inview Oncopanel All-In-One and GATCLiquid Oncopanel All-In-One?

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

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

Can I directly compare results between Inview Oncopanel All-In-One and GATCLiquid Oncopanel All-In-One?

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

What are the limitations of the CNV analysis?

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.

Will all variants in my sample be detected?

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 GATC Biotech’s highly sensitive methods.

Can I use Inview Oncopanel All-In-One for diagnostic purposes?

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

Where do I get my results and in which form are they delivered?

All deliverables can be downloaded via your secure myGATC online account.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Microbiome High Specificity

What kind of samples can be used?

A wide variety of (DNA) samples can be used for microbiome analysis, such as:

  • Environmental samples isolated from all kinds of sources
  • Human samples, such as swabs, faeces, lavage
  • Food samples for quality control and pathogen detection in industrial settings like dairies, breweries, meat-processing and agricultural facilities

How much starting material is required?

For information on the quantity and concentration required for specific library preparation methods please refer to your quote or contact us. For optimum results, we generally require >10 ng of double-stranded, purified DNA for PCR amplification (the total amount depends on the proportion of microbial DNA in the sample). Moreover, the DNA has to be RNA-free with an OD 260/280 of >1.8 and OD 260/230 of >1.9.

What data will I receive?

GATC Biotech will deliver tables (tsv, txt) listing all species present in the analysed sample and the corresponding read counts. In addition, you will receive all of the raw sequencing data.
Operational Taxonomic Units (OTUs), which are classified by BLAST analysis against a curated RDP database, are represented in a table format, including taxonomic classification down to the species level. Only the best hits are considered when assigning OTUs.
A demo data report can be viewed here.

Why is the 16S rRNA gene used for bacterial classification?

The 16S rRNA gene is ubiquitous among all bacteria. Its relatively small size of approximately 1,500 bp and its alternating structure of conserved and hypervariable regions (V1-V9) make the 16S rRNA well suited for identification and phylogenetic analysis of microorganisms. Excellent results are achieved with the V1-V8 primer pair. The option of using the V1-V9 primer pair is also available, but discouraged due to unstable primer attachment to the much smaller V9 region.

What is the necessary coverage for microbiome analysis?

The actual required sequencing depth mainly depends on the desired sensitivity and the complexity of the sample. The current sequencing coverage is about 15,000 reads per run. When in doubt, we recommend determining the required depth of sequencing by performing a trial on a sub-set of samples. The same applies to the experimental setup for pooling up to 10 samples.

Which organisms can be detected?

Phylogenic characterisation and analysis of microbial communities can be performed for various organisms from complex and non-complex food, industrial, environmental and clinical samples. However, not all organisms can be fully resolved using the 16S rRNA gene. The genus Bacillus is one bacterium that cannot be separated through 16S rRNA gene sequencing alone. We can overcome such technical limitations with the use of complementary detection methods with our Customised Solutions service, which offers the same quality as our standardised services.

How do I choose between Inview Microbiome Profiling 2.0 with Illumina and Inview Microbiome High Specificity with Pacific Biosciences?

Both services benefit from optimised PCR conditions with extremely low chimera formation.
The 2 x 300 bp paired-end read mode makes Inview Microbiome Profiling 2.0 ideally suited for sequencing several hypervariable regions at once. Scalable sequencing coverage allows for high sequencing depth as needed. The use of the latest Illumina chemistry provides a high degree of sensitivity and allows for the detection of even very low amounts of taxa in a complex sample. Furthermore, multiplexing makes it possible to process a huge amount of samples in parallel and within in a short turnaround time.
Inview Microbiome High Specificity with PacBio is the method of choice for sequencing of the entire 16S rDNA at the highest possible taxonomic resolution. Long amplicon sequencing allows for a high degree of specificity down to the species level. The Reads of Insert approach results in high-quality data output, where the threshold can be defined beforehand. The high degree of sensitivity of the full 16S analysis with SMRT sequencing technology makes it possible to characterise the microbial community, even at low densities.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Metagenome Advance

What kind of samples can be used?

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

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

How much starting material is required?

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

What data will I receive?

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

What is the necessary coverage for metagenome analysis?

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.

Do you guarantee a certain sequencing output?

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

Which organisms can be detected?

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 GATC Biotech is most experienced in profiling human microbiota, we have also successfully characterised microbial community members from food, industrial and environmental samples.

How do I choose between targeted amplicon sequencing and metagenome sequencing?

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.

What is the difference between Inview Metagenome Explore and Inview Metagenome Advance?

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.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Metagenome Explore

What kind of samples can be used?

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

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

GATC Biotech is particularly well-experienced in DNA isolation and sequencing of stool samples.

How much starting material is required?

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

What data will I receive?

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

What is the necessary coverage for metagenome analysis?

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

Do you guarantee a certain sequencing output?

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

Which organisms can be detected?

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

How do I choose between targeted amplicon sequencing and metagenome sequencing?

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

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

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

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

What is the difference between Inview Metagenome Explore and Inview Metagenome Advance?

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

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Transcriptome Discover

Which Inview Transcriptome package do you recommend for which application?

This depends on many factors, such as tissue type, sample quality, development status, existing references, etc. Please refer to the table shown under Product Specifications to find your optimum InView™ Transcriptome solution. The estimated read numbers shown there refer to human samples. Alternatively, please contact us to discuss your project. In some cases it might be advisable to set up a trial to define how many reads will be required for your specific aim or organism.
 
The Inview Transcriptome Explorepackage gives you an overview of the expressed genes and is well suited to detecting differences between samples.  
 
Inview Transcriptome Discover uses a strand-specific RNA library combined with Illumina’s 150 bp paired-end sequencing and is therefore recommended for the detection of differentially expressed genes, rare and novel transcripts and for discovering splice variants. In addition, the information about the transcript’s orientation allows for a more precise determination of structure and gene expression.  

Do I need a genomic reference sequence?

Not necessarily. However, if you have a reference, please refer to the following recommendations. A clearly defined Ensembl name (e.g., GRCh37 or Rnor_5.0) for the annotated genomic reference sequence has to be provided prior to project start. Alternatively, the genome sequence can be provided in Fasta (along with the corresponding annotation in Gene transfer format (gtf)) or in GenBank format (including annotation). For projects without a reference genome, de novo transcriptome sequencing based on PacBio RS II technology can be added.

Which starting material will be accepted?

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

How much starting material is required?

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

Can RNA isolation be offered?

GATC Biotech 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.

Which starting materials and aims are best suited for performing ribosomal RNA depletion?

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 GATC Biotech. 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.

How efficient is ribosomal RNA depletion?

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.

Should I send my starting material on dry ice or would room temperature be sufficient?

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.

What should I do if my sample fails the entry QC?

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, GATC Biotech will recommend additional pre-processing steps in order to optimise sample quality.

Where and in what form can I obtain my results?

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

What kind of BioIT do you offer?

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

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Transcriptome Explore

Which Inview Transcriptome package do you recommend for which application?

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

The Inview Transcriptome Explore package gives you an overview of the expressed genes and is ideal for detecting differences between conditions or experiments.

Inview Transcriptome Discover uses a strand-specific RNA library combined with Illumina’s 125 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 profound determination of gene structure and expression.

Do I need a genomic reference sequence?

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

Which starting materials will be accepted?

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

How much starting material is required?

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

Is RNA isolation offered?

GATC Biotech can offer RNA isolation as an additional service. We have successfully tested and optimised different protocols for isolation of high-quality RNA from several sample types and sources. 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 and project aims.

Which starting materials and aims are best suited for performing ribosomal RNA depletion?

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 GATC Biotech. 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.

How efficient is ribosomal RNA depletion?

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

Should I send my starting material on dry ice or is room temperature sufficient?

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.

What should I do if my sample fails the entry QC?

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

Where and in what form can I obtain my results?

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

What kind of BioIT do you offer? Do you have demo data?

Affordable high-quality bioinformatics analysis, including data filtering and QC, is optionally available for each package. For more details please refer to Files. 

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Transcriptome Isoform Discover

How is Inview Transcriptome Isoform Discover different from Inview Transcriptome Explore, Advance and Discover?

Inview Transcriptome Isoform Discover is an RNA-seq standardised service that employs PacBio’s SMRT® technology to profile full-length cDNA transcripts. The product is perfectly suited for thorough investigations of isoform diversity and obtaining a full profile of alternative splicing events in eukaryotic organisms.
Inview Transcriptome Explore, Advance and Discover are standardised products that use Illumina’s platforms to sequence a variety of library types and facilitate studies on gene expression, gene regulation and identification of rare or novel RNA species. The products are applicable to any organism of interest and provide analysis of coding and noncoding RNA.

What starting material will be accepted?

Total RNA extracted from eukaryotic organisms can be used to obtain full-length cDNA sequencing libraries.

How much starting material is required?

For exact information on RNA quantity and concentration, please refer to your quote or contact us directly. In general, we require 3 µg of high- quality RNA with an OD 260/280 1.8-2.2 for optimal results.
The quality and quantity of the provided starting material is essential for the success of sample preparation. The use of too little, degraded or contaminated starting material can result in low yields, cause sample preparation to fail, or compromise the quality and amount of the sequencing results.

How many data packages do I need to reach my experimental goal?

ISOFORM DISCOVER - recommended number of packages

Does GATC Biotech offer RNA isolation?

GATC Biotech can offer RNA isolation based on optimised protocols as an additional service. Please consult us to discuss possible sample preparation methods for your individual project requirements.

Should I send my starting material on dry ice or would room temperature be sufficient?

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 must be mailed on dry ice. Alternatively, fresh material can be stabilised in “RNAlater” (from companies like Ambion, SIGMA or QIAGEN) and be sent at room temperature. Please check in advance if the couriers that you are using accept dry-ice shipments.

What should I do if my sample fails the entry QC?

If the quantity or quality of the starting material does not meet the requirements for further processing, we will contact you directly to discuss how to proceed. If possible, GATC Biotech will recommend additional processing steps that could help improve sample quality.

How do I obtain my results and what form will they be in when delivered?

You can download all raw data as well as analysed data in various formats from your secure myGATC online account.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on NGSelect Amplicon

What starting material can be accepted?

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

What kind of quality control do you perform?

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.

What should I do if my sample fails the entry QC?

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, GATC Biotech will recommend additional measures for optimising sample quality.

What kind of BioIT do you offer?

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

Can I use the results for diagnostic purposes?

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

Where do I get my results and in which form are they delivered?

All raw data and analysed data can be downloaded via your secure myGATC online account.

How can I choose between the different NGSelect services?

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

Overview various services of NGSELECT

 

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on NGSelect DNA

What starting material can be accepted?

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

Do I always need a genomic reference sequence?

No, for de novo genome sequencing on the PacBio platform, there is no reference sequence required. For other cases, a clearly defined Ensembl name 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 respective annotation in Gene transfer format (gtf)) or in GenBank format (including annotation).

What kind of quality control do you perform?

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

What should I do if my sample fails the entry QC?

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

What kind of BioIT do you offer?

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

Can I use the results for diagnostic purposes?

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

Where do I get my results and in which form are they delivered?

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

How can I choose between the different NGSelect services?

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

Overview various services of NGSELECT

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on NGSelect Ready-to-load

What starting material can be accepted?

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

What kind of quality control do you perform?

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.

What should I do if my sample fails the entry QC?

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, GATC Biotech will make recommendations for optimisation of sample quality.

What kind of BioIT do you offer?

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

Can I use the results for diagnostic purposes?

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

Where do I get my results and in which form are they delivered?

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

Do you guarantee a certain number of reads?

The actual achieved sequencing output (read length, read quality and number of reads) is directly correlated with the quality of the sequencing library used. GATC Biotech 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.

Is the sorting of c included?

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.

What steps are taken to increase sequencing quality?

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.

How can I choose between the different NGSelect services?

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

Overview various services of NGSELECT

Questions on GATCLiquid Oncoexome

What kind of samples can be processed with GATCLiquid Oncoexome?

For Oncoexome, we accept fresh or retained plasma samples as well as already isolated cell-free DNA. Please refer to our GATCLiquid 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.

How much starting material is required?

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).

What pre-sequencing services are available?

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.

What sequencing coverage should I use?

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.

What kind of quality controls do you perform?

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.

How should I choose between Oncoexome, Oncopanel and Oncotarget?

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.

GATCLiquid Oncoexome 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.

GATCLiquid Oncopanel 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%.

GATCLiquid 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.

How do I obtain my results and what form will they be in when delivered?

All deliverables can be downloaded via your secure myGATC online account.

How soon after the blood draw should I ship the sample?

Blood samples have to be shipped to GATC Biotech 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.

How many samples can I submit?

GATC Biotech accepts any number of samples in multiples of four.

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on NGSelect RNA

What starting material can be accepted?

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

Do I always need a genomic reference sequence?

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

What kind of quality control do you perform?

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

What should I do if my sample fails the entry QC?

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

What kind of BioIT do you offer?

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

Can I use the results for diagnostic purposes?

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

Where do I get my results and in which form are they delivered?

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

How can I choose between the different NGSelect services?

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

Overview various services of NGSELECT

Where should I send my samples?

Eurofins GATC Biotech GmbH
NGS - Laboratory
Jakob-Stadler-Platz 7
78467 Konstanz
Germany

Questions on Inview Microbiome Profiling 3.0

What Species can be detected using Microbiome Profiling by NGS?

Depending on your choice of target regions, all bacterial, archeal and fungal species of which a sequence is published in the NCBI nucleotide database can be identified. There are currently for example thousands of bacterial species with sequence entries in the database. Closely related species with almost identical DNA sequences will not be discriminated and only the genus (or the family) will be reported.

What are the limitations of using Microbiome Profiling by NGS?

• Closely related species are not distinguishable because of sequence identity.
• Degradation of bacterial DNA may occur in highly processed samples or samples with low pH - a species identification is therefore difficult and sometimes not possible.
• The more starting material we receive, the higher the probability to get excellent sequencing results.

What is the level of false positives / negatives?

• Samples with strongly degraded DNA will give the result “no species identification possible”. To avoid false-positive results, a negative control (water) is always co-analysed with the samples.
• With NGS all DNA amplicons that were successfully amplified from the sample DNA will be detected. Due to the high sensitivity of the method, sequences with low presence in the sequence pool or sequences with unexpected sequence lengths have to be removed from the data analysis during the bioinformatical workflow in order to exclude false-positives due to PCR and/or sequencing errors. Species with a fraction below 0.5 % of all assigned reads are not reported.

How should I choose the target regions and the amount of targets to be analysed?

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

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

Can you target also cyanobacteria with your INVIEW Microbiome Profiling 3.0?

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

What quality control do you apply on my samples?

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

What happens if no or only a weak amplicon can be generated from some of my samples: 

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

Questions on Customised Solutions

Which next generation sequencing services does Eurofins Genomics offer?

Genome sequencing

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

Transcriptome sequencing

  • De novo transcriptome sequencing
  • Expression profiling

Resequencing & Amplicons

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

Bioinformatics & Special Services

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

Where should I send my samples for individual NGS projects?

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

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

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

 

GATC Biotech AG
NGS-Laboratory
Jakob-Stader-Platz 7
78467 Konstanz
Germany

What is the principle of the Illumina sequencing technology?

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

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

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

FAQ 9.1

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

FAQ 9.2

 

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

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

Do you have references for next generation sequencing projects?

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

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

How do I provide a reference sequence?

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

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

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

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

Do I own intellectual property of my results?

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

What does coverage sequencing mean?

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

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

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

How do you deliver the data?

You will receive your date from our secure FTP server.

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

 

What coverage is recommended for amplicon sequencing?

It depends on the goal of your experiment:

  • To detect rare variants with a detection limit of 5%, you need 1000-fold coverage
  • If you would like to have a detection limit of 1%, you need 5000-fold coverage, and so on

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

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

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

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

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

Typical bioinformatics services after transcriptome sequencing are:

Bioinformatics analysis: What does clustering mean?

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

Clustering of sequencing reads is often performed to

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

What is a de novo assembly?

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

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

What is the difference between a scaffold and a contig?

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

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

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

What does mapping stand for?

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

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

Do you offer BLASTx and BLASTn analysis?

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

What is the difference between BLASTx and BLASTn?

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

Scroll to top ^^