What is direct sequencing and what is next generation sequencing technology?

First, the meaning is different:

First-generation sequencing: refers to the dideoxy terminal termination method, in which the sequence is read by capillary electrophoresis after amplification, and the amount of data obtained each time is small.

The second generation of sequencing: for high-throughput sequencing, the use of beads or high-density chips while synthesizing and sequencing, representing 454, solexa, solid, high-throughput, can obtain several g of data at a time, relative to the third generation, both still need to amplify the signal by amplification, amplification and then detection.

Second, the role is different:

Sanger sequencing utilizes a DNA polymerase to extend primers bound to a template of a pending sequence. Until incorporation of a strand-stop nucleotide.

Ordinary next-generation sequencing, whole genome or exome sequencing, splits DNA into small fragments and then reads each fragment multiple times (typically thirty times). However, if mutations occur in only 15-20% of cells, thirty reads are not enough to reliably capture them, especially if the mutation affects only one copy of the gene.

Sequencing procedures

Add the appropriate primers, template, 4 DNTPs (including radiolabeled DNTPs, e.g., 32 PDNTP, and DNA polymerase (reverse transcriptase if RNA is used as template) to each of the 4 tubes, and add a concentration of DDNTP (dideoxynucleotide) to each of the 4 tubes.

Primers bound to a single-stranded template (e.g., double-stranded template, to be denatured) are extended from the 5' end to the 3' end under the action of DNA polymerase, and 32P is incorporated into the new synthetic strand with the primer extension. When DDNTP is incorporated, it does not bind to the next DNTP because it does not have a hydroxyl group at the 3' position, thus terminating the chain elongation. DDNTP is incorporated at different locations, resulting in a series of new DNA strands of different lengths.

The above content refers to: Encyclopedia - Dideoxy Chain Termination Method.

Next-generation sequencing technology, also known as high-throughput sequencing technology, is a revolutionary change from traditional Sanger sequencing (known as next-generation sequencing technology). The core idea of next-generation sequencing technology is sequencing while synthesizing, which can sequence hundreds of thousands to millions of DNA molecules in parallel at a time, so it is also called deep sequencing. Next-generation sequencing technology has been widely used in the field of life sciences due to its advantages of simultaneous sequencing of multiple samples and multiple sites and high coverage.

A process that converts a DNA chemical signal into a digital signal that can be processed by a computer.

Sequencing technology has now developed to the third generation, and the basic methods are to read out the DNA sequences one by one through various physical or chemical techniques, such as the bands of first-generation sequencing, the fluorescence of next-generation sequencing, and the current signal of third-generation sequencing. The second-generation Hahashi sequencing is the current mainstream technology.

As shown in the figure above, 4 normal DNA and 4 DIA were added to a large number of single strands to be sequenced for random reaction, and the chain reaction was terminated when DIA. The termination point can be anywhere on the sequence.

Therefore, when the reaction is sufficient, each base of the sequence to be measured has at least one termination point (at the dideoxynucleotide).

Another method of operation is to add the four dideoxynucleic acids separately, so that for each sample of dideoxynucleic acids, the position of the electrophoresis belt is the position of the corresponding base on the sequence. As shown in the figure below.

DNA sequencing is a reflection of one-way sequencing, which is a reflection of DNA sequencing that distinguishes between measurements.

About DAO can be measured about 800bp, if the measured sequence is about 800bp, then a reflection can be measured, that is, the sequencing is completed;

If the sequence is larger than 800 bp, such as 1500 bp, two reactions are required to complete the sequencing, and the sequencing of the two reactions has the same part in the middle, so splicing is required.

If it is PCR, then due to the sequencing principle, the sequence behind the primer cannot be read, in this case, if you do one-way sequencing, then the sequence behind the first primer is not available, and then you need to sequence from the other end, so you can choose the testthrough.