ngs second-generation sequencing steps and processes

1. Introduction to second-generation sequencing 1. Next-generation sequencing (NGS): Also known as high-throughput sequencing, it is a DNA sequencing technology developed based on PCR and gene chips. 2. First-generation sequencing is synthetic termination sequencing, while second-generation sequencing pioneered the introduction of reversible termination ends, thereby achieving sequencing while synthesis. 3. Next-generation sequencing determines the sequence of DNA by capturing special markers (usually fluorescent molecular markers) carried by newly added bases during the DNA replication process.

4. Existing technology platforms mainly include Roche’s 454 FLX, Illumina’s Miseq/Hiseq, etc. 5. In second-generation sequencing, a single DNA molecule must be amplified into a gene cluster composed of the same DNA, and then replicated synchronously to enhance the fluorescence signal intensity to read out the DNA sequence; as the read length increases, the gene cluster replicates. The decrease in cooperativity leads to a decrease in the quality of base sequencing. Currently, the longest read length of the second generation is 600bp of miseq. 6. Second-generation sequencing is suitable for amplicon sequencing (such as the variable regions of 16S, 18S, and ITS), while genomic and metagenomic DNA need to be broken into small fragments using the shotgun method, and bioinformatics methods are used after sequencing. splicing. Shotgun method: A technical method that randomly processes macromolecule target DNA into small fragments of different sizes for sequencing, and assembles these short sequences into target DNA in subsequent bioinformatics analysis. Traditional method: Use restriction endonuclease to cut the restriction endonuclease recognition site on the target DNA to form small fragments. Commonly used methods: Use mechanical methods (such as ultrasonic DNA fragmentation) to form macromolecule DNA into short sequence fragments distributed within a certain length range.

Background and characteristics of second-generation sequencing 1. In 2005, Roche launched the first second-generation sequencer, the Roche 454, and life sciences began to enter the era of high-throughput sequencing. 2. With the launch of the Illumina series sequencing platforms, the price of second-generation sequencing has been greatly reduced, promoting the popularity of high-throughput sequencing in various research fields of life sciences. 3. Features: high throughput, short read length

3. NGS experimental steps 1. Nucleic acid extraction and detection 2. Library construction and library detection 3. On-machine sequencing

Principle: DNA separated from cells is DNA bound to proteins, which also contains a large amount of RNA, that is, ribonucleoprotein. Using the property that DNA is insoluble in 0.14mol/L NaCl solution but RNA is soluble in 0.14mol/L NaCl solution, DNA nucleoprotein and RNA nucleoprotein can be separated from the sample broken cell fluid. After the nuclear protein is separated, impurities such as proteins need to be further removed. There are three methods to remove protein: ① Shake the nuclear protein solution with chloroform containing octanol or isoamyl alcohol to emulsify it, and then centrifuge to remove the denatured protein. When processed in this way, the protein stays between the water phase and the chloroform phase, while the DNA is dissolved in the upper water phase. The DNA sodium salt can be precipitated out with twice the volume of 95% ethanol solution. ② Use detergents such as sodium dodecyl sulfate (SDS) to denature proteins to separate them from nucleic acids. DNA can also be extracted directly from the material. ③ Treat with phenol and then centrifuge to separate DNA+ and protein. At this time, the DNA is dissolved in the upper water phase, and the protein will stay in the phenol layer after denaturation. Aspirate the upper water phase, and add twice the volume of 95% ethanol to obtain a white fibrous DNA precipitate. By repeatedly using the above method to treat the DNA nucleoprotein solution multiple times, impurities such as proteins can be removed more thoroughly and a purer DNA product can be obtained.

Magnetic bead adsorption method, column collection method DNA extraction steps ✱ Cell lysis ✱ Removal of impurities ✱ DNA recovery ✱ Wash and dissolve DNA

2. Library construction and library detection NGS library construction: the process of adding adapter modifications to DNA fragments. Take the kit NEBNext®Ultra™II DNA Library Prep Kit for Illumina® as an example

Specific steps: ① End modification: 1. Use Tap polymerase to fill up the uneven ends 2. And add protruding base A at both ends to generate sticky ends (if Tap enzyme amplification is used, no end modification is required) 3 .Adapters can be added to fragments that produce sticky ends

②Add adapters: 1. The end of the end-modified PCR fragment has a protruding A tail, and the adapter has a protruding T tail. Ligase can be used to add adapters to both ends of the DNA fragment. 2. The NEB linker has a special base U-linked ring structure (which can enhance stability). Therefore, after connecting the linker, the base U needs to be deleted to form a "Y" type linker. 3. The adapter added in the previous step is mainly used as a primer amplification in the subsequent PCR to continue to add the library index and the oligonucleotide sequence complementary to the sequencing platform (in addition, it is also used as the sequencing primer Rd1 SP/Rd2 SP). 4. The reason for this The "Y" type split structure is because each end of the connector is two non-complementary sequences (each end is Rd1 SP and Rd2 SP staggered), the ligase has no selectivity. Each linker is linked to DNA only by protruding T. The "Y" linker ensures that each single sequence is a different sequencing primer, so that different sequencing primers can be connected in subsequent PCR. The tube nucleotide sequence (P5/P7).

③ Magnetic bead purification: The library system after adding adapters contains various enzymes such as polymerase and ligase as well as auxiliary substances. The adapters are also added in excess, and due to the instability of the ends, self-ligated fragments are easily formed, making shotgun purification There may also be large fragments in the fragments, so special magnetic bead (AMPure XP Beads) purification is required to remove large fragments and various magazines, so as to obtain library fragments with successfully added adapters. 1. Principle: Magnetic beads can adsorb DNA fragments through hydrogen bonding and other forces. The magnetic beads themselves do not have the ability to select fragment size, but there is 20% PEG 8000 in the buffer stored in them. The higher the PEG concentration, the more likely it is to adsorb DNA fragments. The smaller the DNA fragment. 2. Note: During magnetic bead purification, the amount of magnetic beads added (actually the amount of PEG added) must be strictly controlled according to different library fragments to achieve fragment selection.

④PCR amplification: 1. DNA fragments with adapters added can be amplified using primers complementary to the adapters. 2. The fragment also needs to add a specific index to distinguish different libraries, and two oligonucleotide sequences (P5/P7) complementary to the sequencer chip.

⑤Second magnetic bead purification: 1. After PCR, the product DNA fragment needs to be separated from impurities such as polymerase, so magnetic bead purification is performed again. 2. Then perform quality testing, including DNA concentration detection, agarose gel electrophoresis and fragment length detection, to complete library construction.