DNB Preparation technology includes DNA single strand circularization and DNB making.
DNA single strand circularization
DNA single strand circularization: double stranded DNA with adapter sequences at the terminal ends is heated to denature and generate ssDNA (single stranded DNA). A splint oligonucleotide with a complementary sequence to both the 5’ and 3’ terminal ends of one strand of the target dsDNA will hybridize to both the 5’ and 3’ terminal ends of the same target ssDNA to form a nicked circle (Figure 1). The nick is then repaired using DNA ligase to form a single stranded circle.
DNA nanoballs are generated by rolling circle amplification (RCA) using the single stranded circle as a template. Various sizes of DNA fragments were amplified to roughly 100 to 1000 copies (Figure 2). DNB concentration can easily be quantified with Qubit measurements before loading onto the sequencing chip. No expensive quantification instrument or reagents are required.The primary benefit of rolling circle amplification (RCA) is the reduction in error introduced during amplification. RCA utilizes a very high-fidelity DNA polymerase, and each amplification uses the original copy of the DNA circle as the template. This makes it almost impossible to have amplification errors in the same position for all 100-1000 copies of a DNB. In addition, RCA technology avoids the exponential accumulation of errors, GC biases and dropouts observed with other amplification methods, such as PCR. All results in greatly improved sequencing accuracy with the DNBSEQ platform
Using a state-of-art semiconductor manufacturing process, a patterned binding site is created on the surface of a silicon chip. The distance between active spots on the chip surface is uniform, and each binding site is only large enough to bind one single DNB. This ensures there is no interference between the fluorescence signals from neighboring DNBs. This results in high sequencing accuracy, high chip utilization, and optimal reagent usage.
DNBs carry a negative charge in acidic conditions due to its phosphate backbone while the slide surface carries a positive charge. This positive and negative interaction is the main driving force behind DNBs loading onto the slide surface. The proprietary loading buffers can further ensure DNBs sticking on the same spot for hundreds of cycles without any compromised signals.
DNBs are optimized so they are the same size as the active sites on the slide surface. This ensures that only a single DNB is loaded onto each active site, which improves effective spot yield.
cPAS Technology: After sequencing primers are hybridized to the adapter region of the DNB, a fluorescently labeled dNTP probe is incorporated with a DNA polymerase (Figure 4). Any unbound dNTP probes are then washed away, DNB Flow Cell is imaged (Figure 4: Imaging), the fluorescence signal is converted to a digital signal, and the base information is determined using MGI's proprietary base-calling software. After the image is taken, a regeneration reagent is added to remove the fluorescent dye and prepares the DNBs for the next cycle.
The sequencing reaction time has been reduced to less than one minute due to significant improvements in sequencing biochemistry and the identification of a superior sequencing polymerase screened from tens of thousands of mutants.
2nd Strand Preparation
After finishing the 1st strand sequencing, the 2nd strand generation primers and a polymerase with strand displacement activity are added to initiate 2nd strand synthesis. The polymerase will extend the new primer until it reaches the original sequenced strand, at which point it will displace the original sequencing strand to form a new single-stranded template. The newly generated 2nd strand is optimized to maximize the length of the strand while ensuring the strand remains attached to the original DNB. After the 2nd strand sequencing primer is hybridized, the same sequencing chemistry is used for 2nd strand sequencing as was used for 1st strand sequencing (Figure 5). The new 2nd strand template has many more copies of insert DNA, which yields a much stronger signal and increased sequencing accuracy for the 2nd strand.
Base Calling Algorithm
Base calls and base call quality is calculated based on the signal intensities from all channels. The relationship between signal characterization and sequencing error is well established based on known data models. Predicted sequencing errors for unknown samples are calculated based on signal characterization. Quality scores are based on phred-33 standard.
MGI has developed a propriety Sub-pixel Registration algorithm, which enables image intensity extraction at the sub-pixel level, and greatly improves base call accuracy.
Our industry-leading technology has dramatically increased data processing speed and accuracy through integration of a GPU accelerated algorithm, optimization of execution efficiency, and real time image analysis and base calling.