High-throughput sequencing, also known as "Next-generation" sequencing technology, allows for the parallel sequencing of hundreds of thousands to millions of DNA molecules at once. This technology is characterized by its shorter read lengths and high throughput, making it a powerful tool in modern genomics.
**Applications of High-Throughput Sequencing Technology**
Sequencing technology has significantly advanced scientific research. With the rapid development of second-generation sequencing, the scientific community has increasingly applied this technology to address complex biological questions. For instance, de novo sequencing of species without a reference genome helps establish a reference sequence, which is crucial for future research and molecular breeding. Whole genome resequencing of species with an existing reference allows scientists to scan and detect mutation sites across the entire genome, uncovering the molecular basis of individual differences.
At the transcriptome level, whole transcriptome resequencing aids in studying alternative splicing, coding sequence single nucleotide polymorphisms (cSNPs), and small RNA sequencing. By isolating specific-sized RNA molecules, researchers can identify new microRNAs. When combined with techniques like chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP), this approach enables the detection of DNA regions and methylation sites that interact with specific transcription factors.
A notable application is targeted resequencing, which combines second-generation sequencing with microarray technology. This method uses microarrays to synthesize oligonucleotide probes that target specific genomic regions, enriching those segments before sequencing. Agilent and Nimblegen are leading platforms for sequence capture, with human exome sequencing being the most widely used. Exome sequencing offers advantages over whole-genome resequencing, including lower costs and less computationally intensive data analysis, while providing more direct insights into biological phenotypes.
Currently, high-throughput sequencing is being widely used to identify candidate genes for diseases. Researchers at the University of Nijmegen used this technique to discover pathogenic mutations in Schinzel-Giedion syndrome, a rare condition associated with severe mental retardation, tumors, and congenital malformations. They sequenced the exomes of four patients using Agilent SureSelect and SOLiD, achieving 43-fold coverage and producing 2.7–3 GB of mapable sequence data per individual. Their focus on shared variants helped narrow down the candidate gene to one.
Similarly, the Baylor College of Medicine Genome Sequencing Center is conducting studies on over 15 diseases, including various cancers, diabetes, and autism, aiming to better understand the genetic causes and effects of mutations. Exome sequencing was recently highlighted as one of Science’s top ten breakthroughs.
**Development Status of Sequencing Technology**
Historically, several sequencing technologies have emerged, including Massively Parallel Signature Sequencing (MPSS), Polymerase Cloning (Plonony Sequencing), 454 Pyrosequencing, Illumina (Solexa) sequencing, ABI SOLiD sequencing, Ion semiconductor sequencing, and DNA nanoball sequencing.
With the advancement of second-generation sequencing, it has become a key tool in solving biological problems. It enables de novo sequencing of species without a reference genome, helping build foundational data for future research. It also supports genome-wide resequencing, allowing the identification of mutations and understanding of genetic variation.
At the transcriptome level, it aids in studying alternative splicing, cSNPs, and small RNAs. When combined with ChIP and MeDIP, it provides insights into gene regulation and epigenetic modifications.
**Summary and Future Outlook**
First-generation sequencing, known for long reads and high accuracy, is ideal for constructing genomes of new species and filling gaps in genome assemblies, but it is costly and labor-intensive. Second-generation sequencing includes technologies like 454, Solexa, and SOLiD. While 454 offers longer reads suitable for de novo sequencing, Solexa provides high throughput at a lower cost, making it ideal for both large and small genomes. SOLiD excels in SNP detection due to its two-base error correction system, though its short reads limit its use in genome assembly.
Third-generation sequencing, based on single-molecule real-time and nanopore technologies, is still under development and not yet commercially available. However, it holds great promise for future applications in genomics.
Overall, high-throughput sequencing continues to revolutionize our understanding of biology and disease, driving innovation in research and medicine.
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