**High-throughput sequencing**
The sequencing protocol is based on the dideoxy sequencing method first described by Sanger et al. (1977). To sequence both ends of each clone insert, each plasmid template DNA plate must be paired with two 384-well cycle sequencing plates. The sequencing reaction uses Big Dye Terminator chemistry version 3.1 from Applied Biosystems, along with standard M13 or commonly used forward and reverse primers. This process is automated using a Biomek FX pipetting station from Beckman. The robotic arm handles the precise transfer of the template DNA into the reaction mix, which includes dideoxynucleotides, fluorescently labeled nucleotides, Taq DNA polymerase, primers, and buffer.
Each template and plate is equipped with unique barcodes, which are tracked by the Biomek FX system to ensure accurate sample handling and prevent errors during the transfer process. A linear amplification step is then carried out in MJ Research Tetrads or 9700 Thermal Cyclers. After amplification, the reaction products are precipitated efficiently at room temperature using isopropanol and can be stored at 4°C or resuspended in water. Once the sequencing instrument is ready, a sample film is automatically generated after scanning the barcode of the reaction plate. The plate is then loaded onto an ABIPrism 3700 or an Applied Biosystems 3730xi DNA Analyzer for electrophoresis. These systems can handle up to 8 runs per day on the ABIPrism 3700 and 12 on the 3730xi, with a setup time of less than one hour.
High-throughput sequencing requires robust automation through a Laboratory Information Management System (LIMS), as described by Kerlavage et al. (1993). At institutions like TIGR, this system covers all stages from initial sequencing to final data tracking. The data is stored in a Sybase relational database, allowing users to trace back from annotated genes to original sequencing traces. The LIMS also includes client/server applications for sample management, data entry, library construction, and sequence processing. Over time, these systems have evolved to incorporate new methods, instruments, and software, becoming more stable and efficient. Key features include automated vector removal, detection of repetitive elements, identification of contaminated clones, and tracking of sample templates.
A user-friendly interface allows daily monitoring of template and sequence quality, ensuring that issues are quickly identified and resolved. Quality control teams work closely with production teams to inspect reagents, monitor template quality, and detect anomalies in the process. They also provide guidance, create standard operating procedures, and ensure consistency across all operations.
**Top 5 Challenges in High-throughput Sequencing**
As gene sequencing becomes increasingly vital in medical and health industries, its application in clinical settings has grown rapidly. Especially with the rise of precision medicine, gene sequencing has become a powerful tool for solving complex biological problems. However, despite significant advancements in technology, many challenges remain.
Over the past decade, high-throughput sequencing has advanced dramatically, with increased capacity and reduced costs. There are now over 10,000 sequencing devices worldwide. Major companies like Illumina have focused on improving ease of use, introducing desktop systems such as NextSeq, MiSeq, and MiniSeq, which reduce manual steps and boot times. Other platforms, like Ion Torrent, have also made strides, with the latest Ion S5 designed to streamline the entire workflow from library preparation to data generation.
Despite these improvements, many challenges persist. One of the most critical is **sample quality**. While testing platforms are often calibrated, real-world samples—especially FFPE (formalin-fixed paraffin-embedded) samples—are frequently problematic. These samples are widely used due to their abundance and clinical value, but the fixation and storage processes cause extensive DNA damage. This damage can lead to failed library construction or inaccurate results if not properly assessed.
Another challenge is **sequencing library construction**, which remains costly in certain applications, such as bacterial genome or low-depth RNA sequencing. While some groups have explored cost-effective homemade solutions, commercial development has been limited. Innovations like 10X Genomics’ Chromium system allow for high-throughput single-cell analysis, offering promising alternatives.
The distinction between **long and short reads** also presents difficulties. While short reads from Illumina platforms are ideal for detecting SNPs and counting transcripts, they struggle with repetitive regions and long structural variations. Long-read technologies, such as those from Pacific Biosciences and Oxford Nanopore, offer better resolution for these challenges. Techniques like linked reads from 10X Genomics help bridge the gap, providing longer-range information without the need for full long reads.
Data analysis is another major hurdle. With the sheer volume of data produced—up to 90 GB per sample or 9 TB for 100 samples—storage and processing become overwhelming. Tools like BAM and VCF files help manage this data, but researchers still face challenges in selecting the best analysis tools among thousands available.
Finally, **clinical interpretation and reimbursement** remain unresolved. Despite the potential of NGS, interpreting genetic variants consistently is difficult, and there is no standardized framework. Additionally, reimbursement for clinical interpretation is nearly nonexistent, making it hard for labs to justify the cost of such services.
These challenges highlight the complexity of high-throughput sequencing and the ongoing efforts needed to make it more reliable, accessible, and practical for widespread use.
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