"Ordinary gene sequencing" should refer to "conventional DNA sequencing", which is a method of sequencing by the Sanger method (that is, the dideoxy method). At present, it is very common to perform automatic sequencing directly with ABI 3730xl, which can basically be done. Read lengths from 600bp to 800bp.
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 was relative to manual sequencing or running flat-plate glue.
However, 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.
The main features of NGS are:
1. High throughput. A RUN can generate 500Mb-600Gb of data.
2. The reading length is relatively short. 454 (about 400-500 bp), llumina (100-250 bp), SOLiD (75-100).
3. The cost of unit data is very low. The cost of sequencing many projects now. Already very low. The cost of bioinformatics analysis has become even more important.
What is high-throughput gene sequencing?The sequencing protocol was based on the dideoxy sequencing method (Sanger et al., 1977). In order to sequence from each of the clone inserts in pairs, each plasmid template DNA plate should be equipped with two 384-well cycle sequencing plates. The sequencing reaction used Big Dye Terminator chemistry version 3.1 (Applied Biosystems) and standard M13 or commonly used forward and reverse primers. The sequencing reaction was established by a Biomek FX (Beckman) pipetting station. The robotic arm is responsible for aliquoting the template sample and mixing it with the reaction solution. The reaction solution contains dideoxynucleotides, fluorescently labeled nucleotides, Taq DNA polymerase, sequence primers and buffer. The template and plate have barcodes and barcode reader tracking on the BiomekFX pipetting station to ensure no errors in template and reaction transfer.
The 30-40 linear amplification step was continuously performed in MJ Research Tetrads or 9700 Thermal Cycler (Ap-plied Biosystems). The reaction product can be precipitated with high efficiency at room temperature with isopropanol, stored or suspended in water at 4 C. If the sequencing instrument is normal, a sample film will be automatically generated for each reaction plate after scanning the barcode of the reaction plate. The plate was then transferred to an ABIPrism 3700 DNA Analyzer or an Applied Biosystems 3730xi DNA Analyzer for electrophoresis. The current multimers and software allow for 8 electrophoresis per day on the ABIPrism 3700 DNA Analyzer and 12 times on the Applied Biosystems 3730xl DNA Analyzer with a commissioning time of less than 1 h.
High-throughput sequencing equipment that runs a large number of jobs in parallel typically requires automated management through a laboratory information management and sample tracking system (LIMS) (Kerlavage et al., 1993). At TIGR, this system includes a complete suite of software from early sequencing to end-of-sequence tracking of library construction. After this processing, the data is saved in a Sybase relational database table.
The database stores and links all of the data collected throughout the genome sequencing process, allowing the user to backtrack the data stream in a variety of ways, backtracking from the annotated genes to the original sequencing trace files of the genes. This system includes client/server applications for sample management, data entry, library management and sequence processing. After years of improvement, combined with new laboratory methods, new instruments and software, this system has matured and stabilized. These integrated applications include automated vector removal, identification and masking of repetitive elements, discovery of contaminated clones and tracking clones, and template information.
In the sequencing process, the quality of the generated templates and sequences can be systematically monitored daily through a user-friendly interface. This ensures that potential problems in the work are quickly discovered and corrected. Typically, quality control/quality assessments (QC/QA) groups apply quality inspection standards together. They are responsible for inspecting and providing reagents to the production team, and detecting the quality of the template, the failure of the investigation and deviation from the normal performance range in the process, monitoring the quality of the data, auditing, identifying areas for improvement, and making control documents (standard operating procedures) to Ensure that these files are format consistent and technically accurate.
The significance of high-throughput sequencingThe birth of high-throughput sequencing technology can be said to be a landmark event in the field of genomics research. This technology dramatically reduces the cost of single-base sequencing of nucleic acid sequencing compared to first-generation sequencing technologies. For example, human genome sequencing at the end of the last century cost $3 billion to decode human life codes, while second-generation sequencing The sequencing of the human genome has entered the era of the 10,000 genome. Such low cost of single base sequencing allows us to implement genome projects for more species to decrypt the genomic genetic code of more biological species. At the same time, large-scale whole-genome resequencing of other species of the species has become possible in species that have completed genome sequencing.
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