In the rapidly evolving landscape of synthetic glycobiology, the development of robust microbial or mammalian cell factories is the cornerstone of successful bioproduction. However, engineering these strains often involves complex genomic modifications, including gene knockouts, site-specific insertions, and the integration of heterologous glycan biosynthetic pathways. Ensuring that these modifications are accurate, stable, and free from unintended "off-target" effects is critical.
CD BioGlyco provides a world-class targeted sequencing-based chassis validation service. Unlike whole-genome sequencing (WGS), which is data-intensive and costly, our targeted approach focuses exclusively on the genomic regions of interest. By utilizing high-depth next-generation sequencing (NGS), we provide researchers with unparalleled resolution to confirm the genetic integrity of their engineered chassis. This service ensures that your synthetic biology projects transition smoothly from the design phase to large-scale production with absolute genomic certainty.
Key Technologies
Hybridization Capture-based Enrichment
We utilize custom-designed biotinylated oligonucleotide probes to "capture" specific genomic loci, such as integrated glyco-pathways or edited loci. This allows for deep sequencing of target regions even in complex or contaminated samples, providing a higher signal-to-noise ratio than traditional methods.
Ultra-Deep Amplicon Sequencing
For smaller targets or point mutation validation, we employ multiplexed polymerase chain reaction (PCR)-based enrichment followed by NGS. This technology allows us to achieve sequencing depths of >1000×, enabling the detection of rare sub-clonal variations or low-frequency off-target mutations that standard sequencing might miss.
Bioinformatic Variant Calling Pipelines
Our proprietary computational suite is tailored for synthetic biology. It compares the sequenced chassis against the in silico design, identifying single-nucleotide variants (SNVs), small insertions/deletions (indels), and structural rearrangements with high precision.
Precision Targeted Sequencing Validation: Securing the Genetic Foundation of Your Bio-Factory
At CD BioGlyco, our targeted sequencing-based chassis validation service is a specialized subset of our chassis strain validation service. We recognize that the "Build" phase of the design-build-test-learn cycle is only as strong as its validation. This service is meticulously designed to support clients working on complex glycoengineering projects where the precision of the host strain is non-negotiable. Our scope covers a diverse range of organisms, from traditional workhorses like Escherichia coli and Saccharomyces cerevisiae to non-traditional chassis such as Pichia pastoris or Chinese hamster ovary (CHO) cells. We focus on the most critical aspects of chassis integrity, including:
Verification of Synthetic Circuits: Confirming the exact sequence and orientation of multi-gene glycan biosynthetic clusters.
Stability Monitoring: Assessing genetic drift over multiple generations or under industrial fermentation conditions to ensure the chassis remains productive.
Off-target Analysis: For genome-editing applications, we screen predicted off-target sites to ensure no detrimental mutations have occurred.
Copy Number Variation (CNV) Analysis: Quantifying the number of integrated gene copies to correlate genetic dosage with glycan yield.
By providing detailed genotypic profiles, we help our clients minimize the risk of "escaped" phenotypes and production failures, ensuring that every engineered cell conforms to its intended design.
Workflow
Project Consultation and Probe Design
Our specialists collaborate with you to define the genomic regions of interest. Based on your in silico design, we manufacture custom probes or primers tailored to your specific synthetic inserts or edited loci.
Sample Preparation and Quality Control (QC)
Clients provide genomic DNA (gDNA) or cell pellets. We perform rigorous QC using fluorometric quantification and capillary electrophoresis to ensure the DNA is of high molecular weight and sufficient purity for library construction.
Targeted Library Construction
Depending on the project scale, we utilize either hybridization capture or ultra-deep amplicon enrichment. The target regions are selectively amplified or captured, and sequencing adapters are ligated to create a high-quality NGS library.
High-Throughput Sequencing
Libraries are sequenced on state-of-the-art NGS technology. We ensure sufficient depth of coverage to identify even the most subtle sequence variations within the targeted chassis regions.
Advanced Bioinformatic Analysis
Our bioinformaticians align the raw data to the reference genome and the synthetic design. We generate a report detailing SNVs, indels, and structural variants, along with a "fidelity score" for the chassis.
Validation Report Delivery
The final output is a detailed technical report. It includes coverage plots, variant tables, and expert interpretations regarding the suitability of the chassis for downstream applications or further engineering.
Publication Data
DoI: 10.1038/s41598-020-74580-1
Journal: Scientific Reports
IF: 3.9
Published: 2020
Results: This study investigates the somatic mutation landscape of breast cancer (BC) in a homogeneous Swedish cohort (61 tumor-normal pairs, 85% ER-positive) via targeted NGS of a 20.5 Mb array covering 765 genes. Significantly mutated genes (SMGs) include PIK3CA (28%), TP53 (21%), and CDH1 (11%), while histone-modifying genes KMT2C and ARID1A together harbor 28% of mutations, distinguishing this cohort from previous studies. KMT2C mutations are mutually exclusive with PIK3CA mutations and often truncate the functional PHD domain. Recurrent copy number aberrations involve CDK10 deletion (80%) and MDM4 amplification (80%). Mutational signatures point to APOBEC deaminase activity and defective DNA mismatch repair. Age-related differences emerge: TP53 mutations are more frequent in younger patients (29% vs. 9%), and CDH23 mutations are exclusive to those <70. These findings highlight the unique genomic features of Swedish BC, emphasizing histone modification gene roles and age-dependent mutational patterns, with implications for personalized therapy.
Fig.1 Mutational signatures in the Swedish BC cohort. (Mathioudaki, et al., 2020)
Applications
Therapeutic Glycoprotein Production
Ensuring the genetic stability of CHO or yeast strains used to produce monoclonal antibodies (mAbs) or erythropoietin, where glycan uniformity is strictly regulated.
Metabolic Pathway Engineering
Validating the successful integration of complex, multi-step pathways for the synthesis of rare sugars, human milk oligosaccharides (HMOs), or specialty polysaccharides in microbial hosts.
Vaccine Development
Confirming the sequence of viral antigens or glycoconjugate components expressed in bacterial or insect cell chassis to maintain immunogenic potency and safety.
Industrial Biomanufacturing
Monitoring chassis performance in large-scale fermenters by verifying that the genetic architecture responsible for high-yield glycan production remains intact under metabolic stress.
Advantages
Unmatched Precision
Our targeted approach achieves significantly higher coverage depth than WGS, allowing for the detection of minor variants (low-frequency alleles) that could impact long-term strain stability.
Customizable Solutions
We don't believe in one-size-fits-all. Every probe set and analysis pipeline is customized to the specific synthetic parts and host organism used in your project.
Expert Insight
Our team consists of PhD-level scientists with deep expertise in both glycobiology and genomics, ensuring that your data is interpreted within the context of biological function.
Cost-Efficiency
By focusing only on the genomic regions that matter, we reduce sequencing and data processing costs, making high-resolution validation accessible for iterative engineering cycles.
Frequently Asked Questions
Customer Review
"The level of detail provided in the validation report was exceptional. We were able to identify a silent mutation in our glycosyltransferase cluster that was affecting our yields, something our standard PCR screening missed entirely."
– G.T., Senior Researcher
"Working with CD BioGlyco allowed us to validate 50 different chassis variants in record time. Their targeted approach saved us thousands in sequencing costs while giving us the confidence to proceed to pilot-scale fermentation."
– Q.T., Director of R&D
"Reliability is key in our industry. The targeted sequencing service provided the rigorous genotypic data we needed. CD BioGlyco is a partner we can trust."
CD BioGlyco is committed to empowering the synthetic glycobiology community with high-precision, cost-effective genomic validation. Our targeted sequencing-based chassis validation service bridges the gap between design and reality, ensuring your engineered strains are as perfect in the lab as they were on the screen. Please feel free to contact us to help you design the perfect validation strategy.
Reference
Mathioudaki, A.; et al. Targeted sequencing reveals the somatic mutation landscape in a Swedish breast cancer cohort. Scientific Reports. 2020, 10: 19304 (2020). (Open Access)
For research use only. Not intended for any clinical use.
Fig.1 Mutational signatures in the Swedish BC cohort. (Mathioudaki, et al., 2020)
