CeGaT Automates NGS Library Preparation for TMB and MSI Analysis

To ensure an even higher sample throughput, CeGaT has decided to automate the protocols for library preparation used for the analyses of Tumor Mutational Burden (TMB) and Microsatellite Instability (MSI). The automation of the NGS libraries allows CeGaT to perform three times the number of samples per run, while almost halving the hands-on time, as CaseStudy shows.

CeGaT is a genetic diagnostics company that is very active in the field of oncology. The company is not only involved in various clinical research projects but also offers numerous services for Next-Generation-Sequencing (NGS). Two of the most popular analyses in precision medicine oncology are the analysis of tumor mutation burden (TMB) and the analysis of microsatellite instability (MSI). The analysis of these two biomarkers is the basis of immunotherapy, which has revolutionized oncology. Biomarkers are necessary to assess the effect of treatment and support the promising development of new immunotherapy combinations.

To increase sample throughput and reproducibility of results, CeGaT has decided to automate two workflows of the next-generation sequencing (NGS) library preparation protocols used for analysis through their in-house somatic tumor and for analysis through WES. The Hamilton Liquid handling platforms provide CeGaT with the flexibility needed to develop and automate NGS’s library preparation workflow.

The automation of the workflows for NGS library preparation significantly increases the efficiency and reproducibility of the assay by

  • allowing the processing of more samples per run,
  • reducing the hands-on time and
  • reducing the probability for errors, thanks to the full traceability of the samples

Find more information in the CaseStudy.

CeGaT is currently working on further automation of new NGS-based assays for oncology and automates further NGS-based analyses in different areas of biology and life science’. For the future, CeGaT wants to expand its portfolio and automate as many methods as possible. Our goal is to provide the highest quality standards for sample analysis while remaining adaptable to the rapidly evolving NGS methods. It is crucial for us to always use the most modern technology and keep developing ourselves to offer you the most innovative and best sequencing technology in the long run and thus be the best choice for you.

Innovation is essential for us. We are continuously expanding our product portfolio to apply the latest developments in genetic diagnostics.

CeGaT Researches on Best Practice for Microbiome Analysis

CeGaT addresses the urgent need for standardized microbiome analysis workflows by comparing three commonly used sequencing library protocols.

So far, a scientific “best practice” consensus for shotgun metagenomics has not been found. Data quality, analysis accuracy, and repeatability of studies strongly vary, depending on the wet lab protocol and analysis workflow chosen by the scientist. Standardized, highly accurate microbiome analysis workflows could contribute to study comparability. For providing more standardization, CeGaT tested key quality parameters of three commonly used sequencing library protocols for shotgun metagenomics:

•             Protocol A: Illumina Nextera DNA Flex (input: 100 ng)

•             Protocol B: Illumina Nextera XT DNA (input: 1 ng)

•             Protocol C: Down-scaled Illumina Nextera XT DNA (input: 0.1 ng).

The different libraries were sequenced using the same parameters. The generated data was mapped against a database. The analysis focused on the correct representation of a microbial community’s composition, detectable diversity, and repeatability.


To test the performance of the protocols, we compared the three library preparations using the same standard. We used a commercially available microbial community standard that consists of DNA from ten microbial species at a known composition.

All protocols were able to capture the microbial species present in the standard. However, regarding the correct representation of the community composition (i.e. the theoretical relative sequence abundance of the detected microbial species) the protocols revealed differences. The theoretical composition was most accurately determined with protocol A.

Detectable microbial diversity and repeatability

For the investigation of repeatability and detectable diversity, we used a complex microbial DNA pool, generated from ten individual stool samples. We performed three library preparations for each protocol using this complex microbial DNA pool.

All protocols were able to detect the most common gut microbiome representatives and covered a wide range of different microbes. To evaluate the microbial diversity within a sample, we calculated the species richness and evenness as well as the Shannon Diversity Index. Each parameter was slightly higher with protocol A.

The highest repeatability was also shown by Protocol A, though the difference was slight. In comparison with protocol B and C, protocol A had the lowest variation between replicates among the most abundant taxonomic units.


Our detailed comparison of three different library preparation protocols for shotgun metagenomics revealed that protocol A achieved the best results for both the microbial community standard and the complex stool samples. Based on these results, protocol A represents an accurate standard protocol and is highly suitable for metagenomic profiling. Furthermore, protocol C, which showed only a slightly lower accuracy than protocol A, seems to be a good alternative for extremely low concentrated DNA samples due to its very low input-requirements.

Further information on the study and the results can be found in our Tech Note.

Your Science. Our Sequencers.

Our mission is to support scientists like you to accomplish your project, regardless of its scope. With rapid advancements in sequencing technology and capacity, we believe that genome sequencing as the gold standard for genetic analyses should be available to every scientist committed to the wide field of biology. We are your first choice genome sequencing provider, ready to meet your demands and to provide you with a genomic dataset – fast, affordable, and with outstanding service.

Genome sequencing for everyone – nothing can stop you now

To underscore our commitment to the scientific community, we have increased our sequencers fleet to five NovaSeq 6000s. This enables us to sequence additional 9000-14000 genomes per year. With the capacity gained, we can offer high-quality sequencing at competitive prices – also for smaller batches. We hence make genome sequencing accessible for all research groups, with sequencing demands in any quantity. 

Interpreting genomic data is vital – good thing that we have ten years of experience 

The sequencing of the whole genome provides a complete picture of an organism’s set of genetic information, which is especially important for rare disease and cancer research. Still, its interpretation is limited and remains challenging. As a pioneer in genetic diagnostics, we use our ten years of experience in solving cases to create and further develop new methods in genetics analyses. For more than one decade, we have successfully supported numerous projects from start to finish. We know what it takes to achieve the best results.

Tomorrow’s genetic research and diagnostics available today 

We are continuously growing and expanding to strengthen our position as your partner of choice for research projects – today and tomorrow. We are interested in developing solutions together with you, and we are your approachable partner with extensive, dedicated customer service. We use proven technology and adhere to the highest standards. All project steps are carried out in-house and under constant scientific supervision to maintain the best results. CeGaT is accredited according to CAP/CLIA and DIN EN ISO 15189, hence meeting high international laboratory standards. We are a privately held, independent company that only delivers fast, reliable, and trusted quality made in Germany.

Find more information about genome sequencing here.

More Information about Sequencing

Tumor Mutational Burden and Other Biomarkers Help to Assess the Efficacy of Immunotherapies

Immunotherapy has revolutionized the treatment of many types of cancer by taking advantage of the immune system’s ability to fight cancer cells. In the last few years, a growing number of immunotherapy-based clinical trials were launched to increase success rates further. Specific biomarkers help to stratify patients for the best treatment choice, and also to support the promising development of new immunotherapy combinations.

The center for Dermatooncology at the University hospital Tübingen conducted a prospective biomarker study (Forschner 2019) in cooperation with CeGaT GmbH. This study aimed to identify reliable biomarkers, which predict the patient’s response to anti-CTLA-4 and anti-PD-1 immunotherapy.

Tumor mutational burden (TMB) is a biomarker that has been described as particularly useful for immunotherapy and was thus assessed as part of this study. TMB refers to the number of mutations present in a cancer patients’ tumor and is quantified as mutations per million base pairs (mut/Mbp). Furthermore, repetitive liquid biopsies were collected to determine the concentration of circulating tumor DNA (ctDNA) during treatment. Several studies have shown that ctDNA levels are associated with treatment response and prognosis and could therefore serve as non-invasive predictive markers.

Within this study, we demonstrated that patients with a high TMB (>23.1) showed a significantly better response to therapy and, consequently, a better outcome compared to patients with TMB-low or TMB-intermediate tumors. Similarly, undetectable levels of ctDNA, or a >50% decrease of the cell-free DNA (cfDNA) concentration at the time of first follow-up (3w post treatment initiation) was significantly associated with therapy response and better overall survival.

The results of this study strengthen the increasing importance of the analysis of biomarkers such as TMB, ctDNA, or cfDNA in the prediction and early assessment of the effectiveness of immunotherapies.


CeGaT Research and Pharma Solution offers different methods of TMB analysis to meet the most diverse study requirements


At CeGaT, the TMB score can be evaluated based on different sequencing approaches. The best choice for a specific project depends on the underlaying scientific question and availability of patient material. Typically, the TMB score is assessed through whole-exome sequencing of the tumor tissue, as well as the corresponding normal tissue (e.g. blood sample). By comparing the variants detected in both samples, we can discriminate between tumor specific (somatic) mutations, and mutations present in every tissue of the patient.

However, recent studies have shown that the TMB score can also be effectively estimated if only a panel of genes gets sequenced, provided the sequencing panel covers a genomic region of at least 1,5 Mb (Buchhalter 2018). Thus, for many cases, it is not only sufficient but even more expedient to analyze a selected panel of genes. Therefore, we make use of our CeGaT somatic tumor panel which comprises more than 700 selected, cancer-related genes.

Clinical samples are very precious material and availability is often limited. A comparison of tumor and normal tissue might not always be possible. To meet this challenge, CeGaT uses the solution provided by Illumina, in which TMB score is evaluated based on tumor tissue analysis only: TruSight Oncology 500 (TSO500) comprises the next-generation sequencing based analysis of 523 cancer-relevant genes and their standardized bioinformatic assessment. No normal tissue is required.

For the most comprehensive results, CeGaT’s customers always receive information about the tumor’s microsatellite instability (MSI) in addition to TMB analysis – regardless of whether it is based on TSO500, whole-exome sequencing, or CeGaT somatic tumor panel sequencing. Besides TMB, MSI is another important biomarker for the patient’s response to immunotherapy.

Contact us in order to find out which TMB product is the best choice for your study.


Combine your TMB analysis with the following services offered by CeGaT Research and Pharma Solutions:

  • Detection and sequencing of certain immune subpopulations (e.g. CD4+, CD8+, PD-1+ T cells)
  • Detection of (neo)antigen-specific T cells
  • Microbiome analysis
  • T cell receptor (TCR) sequencing
  • Human leukocyte antigen (HLA) analysis
  • Transcriptome sequencing

Further information can be found here.


Forschner A, Battke F, Hadaschik D, Schulze M, Weißgraeber S, Han CT, Kopp M, Frick M, Klumpp B, Tietze N, Amaral T, Martus P, Sinnberg T, Eigentler T, Keim U, Garbe C, Döcker D, Biskup S. Tumor mutation burden and circulating tumor DNA in combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma – results of a prospective biomarker study.
J Immunother Cancer. 2019 Jul 12;7(1):180. doi: 10.1186/s40425-019-0659-0.

Buchhalter I, Rempel E, Endris V, Allgäuer M, Neumann O, Volckmar AL, Kirchner M, Leichsenring J, Lier A, von Winterfeld M, Penzel R, Christopoulos P, Thomas M, Fröhling S, Schirmacher P, Budczies J, Stenzinger A. Size matters: Dissecting key parameters for panel-based tumor mutational burden analysis. Int J Cancer. 2019. Feb 15; 144(4):848-858. doi: 10.1002/ijc.31878.

CeGaT erweitert Leistungsspektrum für Forschung und Pharmabranche

Die CeGaT GmbH baut die eigene Unternehmenssparte für Sequenzierdienstleistungen aus: Mit „Research and Pharma Solutions“ bietet die weltweit operierende Firma individuelle Lösungen für Fragenstellungen aus Forschung und Pharmabranche an.  

Das Tübinger Unternehmen, das primär als Anbieter genetischer Diagnostik gegründet wurde, erweitert stetig Expertise und Angebotsportfolio im Bereich der Auftragssequenzierung. Letzteres umfasst mittlerweile Dienstleistungen, die über den reinen Sequenzierservice hinausgehen, wie etwa Immunomoritoring, Liquid Biopsy und Tumormutationslastanalysen. Fortlaufend wird darüber hinaus in die neusten Technologien investiert. Mit drei eigenen NovaSeq 6000 können innerhalb kürzester Zeit große Mengen Probenmaterial analysiert werden. Durch die Kombination von modernstem Equipment und menschlichem Know-How unterstützt CeGaT zahlreiche Wissenschaftlerinnen und Wissenschaftler erfolgreich bei ihrer Arbeit.

Mit der Neupositionierung reagiert die Firma auf die steigende Nachfrage und richtet sich zudem verstärkt an Kundschaft aus der Pharmabranche. In langfristig angelegten Partnerschaften wird gemeinsam die beste Strategie zur Durchführung von Projekten, wie beispielsweise klinische Studien, erarbeitet.

Dr. Jiri Ködding, Director Operations, sieht in der Umgestaltung einen wichtigen Schritt: „Unsere Vision ist es, CeGaTs Position als Partner der Wahl für Forschungseinrichtungen und Pharmaunternehmen auf der ganzen Welt zu untermauern. Längst machen wir mehr als nur reine Sequenzierungen. Der neue Name trifft besser, wofür wir stehen: Überzeugende individuelle Lösungskonzepte für wissenschaftliche und pharmazeutische Fragestellungen.“

Mehr Informationen zum Portfolio von Research and Pharma Solutions finden Sie hier. CeGaT legt bei der Realisierung von Forschungsvorhaben größten Wert auf beste Qualität. Daher übernimmt die Firma die strengen Anforderungen der genetischen Diagnostik auch für Aufträge aus der Wissenschaft und dem Pharmabereich. Um das hohe Niveau und schnelle Durchlaufzeiten gewährleisten zu können, finden alle Schritte im eigenen Haus statt. Entlang des ganzen Prozesses, vom Design bis zum Abschlussbericht, steht den Kundinnen und Kunden eine qualifizierte Ansprechperson aus der Wissenschaft oder Medizin zur Verfügung. Die CeGaT ist nach CAP, CLIA und DIN EN ISO 15189:2014 akkreditiert.