The third generation sequencing technology is PacBio’s SMRT and Oxford Nanopore Technologies Nanoporous Single Molecular Sequencing Technology. It is characterized by single-molecule sequencing, which does not require PCR amplification.

Mass spectrometry is an analytical method for measuring the mass-charge ratio of ions. The first-order mass spectrometry mainly gives the molecular weight of the target compound. The first-order spectrum of GC-MS can be qualitatively analyzed. LC-MS can be used for simple molecular weight determination. Secondary mass spectrometry can identify some fragments of the target and analyze the structure of the target.

In protein sequencing, the first-order mass spectrometry-bound peptide mass fingerprint (PMF) can preliminarily predict the type and sequence of proteins. The basic principle of PMF is to cut the protein directly from two dimensional electrophoresis gel or cut it onto the PVDF membrane and cut it down. After enzymatic hydrolysis, the peptide segment is obtained, and then the PMF of these peptides is obtained by mass spectrometry, that is, the peptide mass fingerprint is obtained. Because the amino acid sequence of each protein is different, when the protein is enzymatically hydrolyzed, the sequence of the peptide fragments is different, and the mass number of the peptide mixture has certain characteristics. Proteins and nucleic acid sequence libraries can be identified by using the measured quality of peptide segments, combined with appropriate computer algorithms. However, this method can not be used for direct sequencing, it must rely on a large number of database information for comparison, and the accuracy is limited.

Comparison of Sequencing Techniques:

1. Purpose. Both methods are for sequencing.

2. Object. They are all biological cell products. Three generations of sequencing objects are single-cell DNA fragments, which need not be amplified by PCR to analyze single nucleotide species. Mass spectrometry sequencing objects are polypeptide fragments of proteins to analyze single amino acid species. A kind of 3. Results. The sequence of the target fragment was obtained. DNA or RNA sequences were obtained by three generations of sequencing, and polypeptide sequences were obtained by mass spectrometry. The two sequences can be converted to each other for verification.

3. Result. The sequence of the target fragment was obtained. DNA or RNA sequences were obtained by three generations of sequencing, and polypeptide sequences were obtained by mass spectrometry. The two sequences can be converted to each other for verification.

4. Principle. Three generations of sequencing are based on the principle of base complementary pairing, while synthesis and sequencing. Mass spectrometry sequencing is based on the difference of charge-mass ratio of different ion fragments.

5. Detection methods. Three generations of sequencing use fluorescence signal or electric signal detection, different nucleotides correspond to different signals, different signal time can distinguish the type of base modification; Mass spectrometry sequencing through charge-mass ratio data calculation and analysis, there are some specific rules, formulas, but also some insurmountable error factors.

6. Read long. The third generation sequencing has a high reading length of 3000 BP and a sequencing speed of about 10 bp/s. Mass spectrometry sequencing can only target small fragments of polypeptides, about 20 amino acids.

7. Modification. Modification can be detected. Three generations of sequencing were used to detect base modification by the time of nucleotide synthesis, and mass spectrometry was used to analyze the modification of amino acids by charge-mass ratio.

8. Accuracy. The accuracy of three generations of sequencing is about 85% in single sequence and 99.9999% in repeat sequence. The accuracy of mass spectrometry is higher, but some amino acids can not be distinguished by charge-mass ratio.

Series mass spectrometry can be directly used to determine the amino acid sequence of the peptide segment. The process is to select the mother ion from the peptide segment produced by the first mass spectrometry and enter the second mass spectrometry. After collision with inert gas, the peptide segment is broken along the peptide chain. The sequence of the peptide segment is inferred from the difference of the mass number of the obtained peptide segments. The obtained mass spectrometry data can be analyzed either by software provided by the instrument or by hand.