The Past and Present of High-throughput Sequencing

The term "high-throughput sequencing" I think most people won't be unfamiliar, because it is now a hot topic, but how much do you know about high-throughput?

The emergence of all things is inextricably linked, and it is impossible to appear out of thin air. The same is true for high-throughput sequencing. With the advancement of science and technology, people's exploration of microorganisms in the environment has never been terminated. Previously relying on traditional plate-pure culture methods, however, as many as 99% of the microorganisms could not be cultured and identified under the existing experimental conditions. Therefore, high throughput antibody production sequencing technology based on non-culture methods has emerged.

Differences between high throughput monoclonal antibody generation and general sequencing

"Ordinary gene sequencing" should refer to "conventional DNA sequencing", which is a method of sequencing by the Sanger method. It is very common to perform automatic sequencing directly with ABI 3730xl, which can basically achieve a read length of 600 bp to 800 bp. The concept of high-throughput sequencing is actually a relative concept. In 2000, sequencing on instruments such as 3700 and MegaBace was also high-throughput sequencing, which is relative to manual sequencing or running flat-plate glue. After 2005, high-throughput sequencing will refer to second generation sequencing, 454, Solexa (later changed to Illumina) and SOLiD and other second-generation sequencing, than the first generation of sequencing such as 3730. It has been improved by tens of thousands of times, even hundreds of millions of times, so it is called high-throughput sequencing.

High-throughput sequencing technology (also known as next-generation sequencing technology) is a technology developed in recent years, which can sequence hundreds of thousands to millions of DNA molecules in parallel, and is highly efficient. With the advancement of technology, high-throughput sequencing technology is gradually being updated.

The past

Sanger method First generation sequencing technology (dideoxynucleotide end termination method): A DNA polymerase is used to extend primers bound to a template of a sequence to be determined until a chain termination nucleotide is incorporated. Due to the high cost, slow speed, low throughput, etc., it is gradually eliminated.

Present

Second-generation sequencing technologies (mainly including Roche 454 pyrosequencing, Illumina Solexa sequencing and ABI SOLID sequencing), each with the following advantages and disadvantages:

Roche 454 Pyrosequencing Technology - relies on bioluminescence for DNA sequence analysis.

Advantages: read sequence length (400 bp), de novo sequence can be performed;

Disadvantages: The number of duplicate bases cannot be determined.

Illumina Solexa sequencing technology - the principle is to reversibly terminate the chemical reaction.

Advantages: highly automated system, many read fragments, suitable for sequencing of a large number of small fragments (microRNA, lncRNA, etc.);

Disadvantages: The read sequence is short and not suitable for de novo sequence.

ABI SOLID sequencing technology - the core is the ligation reaction of four fluorescently labeled oligonucleotides.

Advantages: very high accuracy is read twice per base, especially for SNP detection; system flexibility allows for pooling of samples and segmentation of sequencing regions;

Disadvantages: The read length is limited by the connection reaction.

The above three sequencing methods have their own advantages and disadvantages. Currently, Illumina's sequencing technology is widely used in scientific research.

In order to meet the increasingly prominent research needs, the third generation sequencing technology (single molecule real-time DNA sequencing technology, sequencing process without PCR amplification) is also available: mainly for single-molecule fluorescence sequencing (SMRT) and nanopore sequencing (nanopore molecule).

Single-molecule fluorescence sequencing: a technique that relies on DNA polymerase-based side-synthesis sequencing, with a read length of up to 100,000 bp;

Nanopore Sequencing: A technical method that relies on exonuclease-based electrical signal sequencing with unlimited read length.

At present, due to the high cost, the single-reading long error rate is high, the bio-information analysis software is not rich enough, and the data accumulation is small, etc., and has not been widely used.

Applications

Transcriptome sequencing (RNA-Seq): study cell performance and function;

Methylation sequencing: epigenetic marker information;

Exome-Seq: study directed-enriched DNA;

Chromatin immunoprecipitation-depth sequencing (ChIP-seq);

Genomic sequencing

Digital gene expression profiling;

Sequence Capture technology: Combines chip and deep sequencing, captures the fragment to be tested with a chip probe, and analyzes the nucleic acid sequence using deep sequencing technology.