NGS006 illumina sequencing
The UWM MGI sequencer adopts the advanced DNBSEQTM sequencing core technology, which includes DNA single-strand cyclization, DNB preparation, Patterned Array, DNB loading, cPAS (Combinatorial Probe Anchor Synthesis), double-end sequencing, CoolMPS, as well as fluidic and optical technologies. Anchor Synthesis, cPAS (Combinatorial Probe Anchor Synthesis), bipartite sequencing, CoolMPS, as well as fluidic and optical detection technologies and base recognition algorithms.
The single-stranded circular DNA is used as a template for Rolling circle amplification (RCA) under the action of DNA polymerase, and the single-stranded circular DNA is amplified to approximately 500 copies of the amplified product called DNB. RCA-based linear amplification effectively avoids the problem of exponential accumulation of PCR amplification errors, thus greatly improving the accuracy of sequencing
Double-stranded DNA (dsD) with junctions is amplified into DNB, which is called DNB. Stranded DNA (dsDNA) with a junction sequence, through high temperature denaturation to form single-stranded DNA (single-stranded DNA, ssDNA), cyclization primer and ssDNA complementary pairing of the two ends of the ssDNA, in the ligase catalyzed by the ssDNA head and tail connected to form a single-stranded ring DNA.
By RCA amplification, the 1copies of the original ssCirDNA library molecules can be amplified to obtain about 500copies
Using advanced semiconductor precision processing, an array of binding sites (spot diameter of about 200nm) is formed on the surface of the modified silicon chip, which realizes the adsorption of the DNA nanorods (about 220-240nm) in a regular arrangement. The uniform spacing of the modified sites on the array chip (~700nm) and the fixation of only one DNB at each site ensures that the light signals from different nanospheres do not interfere with each other, which not only ensures sequencing accuracy, but also improves the utilization efficiency of the sequencing chip
The DNBs are negatively charged under acidic conditions, and are loaded onto the chip through the interaction of positive and negative charges with the assistance of a surface activator. The process of loading DNBs into positively charged activation sites (~200 nm in diameter) on the chip is called DNB loading, and the diameter of DNBs is slightly larger than that of the activation sites on the chip, which avoids multiple DNBs binding to the same site
The DNBs contain ~500 DNA fragment copies, which means that they can anneal to 500 Read1 primers for one-strand cPAS sequencing. one-strand cPAS sequencing.
Take the UW Sequencing Reaction Universal Kit as an example (cyclization portion)
Use double-stranded DNA fluorescence quantification, such as the Qubit? dsDNA HS Assay Kit or the Quant-iT PicoGreen? dsDNA Assay Kit, and follow the instructions for quantification to quantify the PCR purified product. The molar volume of the final PCR product is required to be ≥1 pmol, please refer to the following formula 1 for calculation, e.g.: the interrupted product with a primary band of 300 bp, the main fragment size of the PCR product is 384 bp, and the yield should reach 250 ng. If you need to mix multiple samples for sequencing, it is recommended to design a mixing protocol according to the rules of MGIEasy DNA Adapters manual (refer to Appendix B). If you need to mix multiple samples for sequencing, it is recommended to follow the rules of the MGIEasy DNA Adapters manual to design a mixing scheme (refer to Appendix B), and mix the samples of different Adapters after quantification, the total amount of samples after mixing should be 1 pmol, and the total volume should be ≤48 μL.
The mass of 1 pmol of PCR product should be ng = (DNA primary fragment size/bp)/1000bp×660ng
The PCR products were analyzed by Bioanalyzer, Tapestation (Agilent Technologies); LabChip? GX, GXII, GX Touch
(PerkinElmer); Fragment AnalyzerTM (Advanced (PerkinElmer); Fragment AnalyzerTM (Advanced Analytical) and other devices based on the principle of electrophoretic separation for fragment distribution detection of PCR purified products.
- During step 3.10.3, prepare the digestion reaction solution on ice
Use the Qubit? ssDNA Assay Kit to quantify the PCR purified product according to the instructions of the quantification kit. The final requirement is that the digested product yield (ssDNA)/PCR input (dsDNA) is ≥7%. For example, for a break product with a 300 bp primary band and a PCR product with a primary fragment size of 384 bp, 250 ng of the product should be used for cyclization, and the digest yield should be at least 17.5 ng.
- Initial library DNA concentration ≥ 2 fmol/?L. The library should be quantified with the Qubit? ssDNA HS Assay Kit and the Qubit? Fluorometer. Fluorometer to quantify the library, and calculate the input volume based on the quantification results.
-Note: Input volume V (μL)=N×330 g/mol×40 fmol/(1000×1000×C)
-N is the number of nucleotides (total fragment length of the library), and C is the concentration of the library in ng/μL
.