The process begins with double-stranded DNA that contains adapter sequences at both ends. We apply heat to this DNA, causing it to 'denature' or separate into two single-stranded DNAs (ssDNA). A specially designed molecule, called a 'splint oligonucleotide', plays a crucial role next. This molecule has a sequence that matches both ends of one of the separated strands, allowing it to bind or 'hybridize' to these ends. When it does so, it creates a structure known as a 'nicked circle'. Finally, an enzyme called DNA ligase is used to repair the 'nick' in this circle, transforming it into a seamless, single-stranded circle. This carefully controlled process, part of our DNA Nanoball Preparation technology, is one of the reasons you can place your trust in our DNA sequencing capabilities.
DNA Nanoballs (DNBs) are synthesized using a technique known as Rolling Circle Replication (RCR). In this process, a single-stranded DNA circle serves as the template. DNA fragments of varying lengths are amplified to yield roughly 300 to 500 copies. Interestingly, quantifying the concentration of DNBs prior to loading onto the sequencing chip is straightforward with Qubit measurements, eliminating the need for costly quantification instruments or reagents. A significant advantage of RCR lies in its ability to minimize errors introduced during amplification. RCR employs a high-fidelity DNA polymerase, ensuring that each amplification cycle uses the original DNA circle as the template. This mechanism significantly reduces the probability of encountering identical amplification errors across the 300-500 copies of a DNB. Furthermore, RCR helps bypass the issues often associated with other amplification methods such as PCR, including the exponential accumulation of errors, GC biases, and dropout events. As a result, RCR significantly enhances sequencing accuracy on the DNBSEQ platform, reaffirming MGI's commitment to providing precise and reliable DNA sequencing solutions.
DNA Nanoballs (DNBs) inherently carry a negative charge under acidic conditions, a characteristic brought about by their phosphate backbone. On the other hand, our slide surface is specially designed to carry a positive charge. The natural interplay between these opposing charges serves as the primary mechanism enabling DNBs to adhere firmly to the slide surface. To ensure the DNBs' sustained positioning over hundreds of cycles, we utilize specially developed loading buffers. These buffers maintain the integrity of the signals throughout the process, preventing any compromise in data quality. Moreover, we've fine-tuned the DNBs to match the size of the active sites on our slides. This precision ensures each active site accommodates only a single DNB, maximizing effective spot yield and enhancing overall sequencing efficiency. This careful orchestration of DNB size and slide design is part of our commitment to offering robust and accurate DNA sequencing solutions to scientists and researchers.
cPAS (Combinatorial Probe-Anchor Synthesis) Technology: This technique commences once the sequencing primers bind to the adapter region of the DNA Nanoball (DNB). At this point, a DNA polymerase introduces a fluorescently tagged dNTP probe to the mix. Any unbound dNTP probes are carefully rinsed away before the DNB Flow Cell is imaged. This image captures the fluorescence signals, which are then converted into digital data. The base information, or DNA sequence, is subsequently determined using MGI's proprietary base-calling software. Following the imaging phase, a regeneration reagent is introduced. This agent effectively removes the fluorescent dye and readies the DNBs for the next sequencing cycle. Notably, we've significantly reduced the sequencing reaction time to less than one minute. This substantial improvement is attributed to enhancements in our sequencing biochemistry and the discovery of a superior sequencing polymerase. This particular polymerase was identified from an extensive screening process involving tens of thousands of mutants, showcasing our commitment to delivering the most efficient and accurate DNA sequencing process for researchers and scientists.
Once the sequencing of the first strand is complete, we initiate the synthesis of the 2nd strand. This is accomplished by introducing primers specifically designed for 2nd strand generation and a special type of polymerase that exhibits strand displacement activity. The polymerase elongates the new primer until it encounters the original sequenced strand. At this point, it displaces the initial sequencing strand, thus creating a new single-stranded template. We've optimized this second strand to maximize its length while ensuring its attachment to the original DNA Nanoball (DNB). Upon hybridizing the 2nd strand sequencing primer, the same sequencing chemistry used for the first strand sequencing is employed again. It's worth noting that the newly generated second strand template contains more copies of the insert DNA. This results in a significantly stronger signal and enhances the sequencing accuracy of the second strand. The design of this process exemplifies our dedication to achieving optimal sequencing fidelity.
Our sophisticated base-calling algorithm calculates base calls and their respective quality based on the signal intensities captured across all channels. The correlation between signal characterization and sequencing errors is well understood and grounded in established data models. From this, we predict sequencing errors for unknown samples, which are calculated according to their signal characterizations. All quality scores adhere to the recognized phred-33 standard. To improve base call accuracy, MGI has engineered a proprietary Sub-pixel Registration algorithm, allowing for image intensity extraction at a more granular, sub-pixel level. This unique approach greatly enhances the precision of base call determination. Our cutting-edge technology, inclusive of a GPU-accelerated algorithm, has significantly elevated both the speed and accuracy of data processing. Through the optimization of execution efficiency and real-time image analysis and base calling, we ensure our technology stands at the forefront of the industry.