NGS workstation operation tips

In optimizing the operation process of NGS (Next Generation Sequencing) workstation, it is crucial to master a series of sophisticated operating skills, which span the careful processing of samples, the rigor of library construction, and the precise control of the sequencing process. As well as multiple dimensions such as in-depth mining of data analysis. This article will provide some operational tips and suggestions on key steps, hoping to help you improve the efficiency of your experiment and the accuracy of your results:

1. Sample processing

1. Sample selection and preparation:

1> Ensure that the sample is of good quality and avoid using degraded or contaminated samples.

2>Choose the appropriate sample type (such as blood, tissue, cells, etc.) and collection method according to experimental needs.

3>Properly preserve and transport samples to avoid the introduction of external contamination during collection and processing.

2. Nucleic acid extraction and purification:

1>Use a high-quality nucleic acid extraction kit and follow the extraction steps and conditions recommended by the manufacturer.

2> During the extraction process, pay attention to controlling the operating time and temperature to reduce the degradation and loss of nucleic acids.

3> Test the purity and concentration of the extracted nucleic acid to ensure that it meets the requirements of subsequent experiments.

2. Library construction

1. Fragmentation processing:

1>Choose appropriate fragmentation methods and conditions according to the requirements of the sequencing platform.

2>Control fragmentation time and intensity to obtain evenly distributed DNA fragments.

2. End repair and joint connection:

1> Ensure that the ends are completely repaired to avoid uneven ends.

2>Use high-quality adapters and ligases to ensure adapter ligation efficiency.

3> During the adapter connection process, pay attention to control the reaction time and temperature to avoid self-ligation of the adapter or non-specific connection.

3. Library amplification and quality control:

1>Perform appropriate amount of PCR amplification based on library concentration and sequencing requirements.

2> During the amplification process, pay attention to controlling the number of amplification rounds and conditions to avoid over-amplification leading to a reduction in library complexity.

3> Conduct quality control testing on the amplified library, including testing for concentration, fragment size, distribution and other indicators.

3. Sequencing process

1. Sequencing platform selection:

1>Choose an appropriate sequencing platform based on experimental requirements and the performance characteristics of the sequencing platform.

2. Sequencing reaction settings:

1>Follow the sequencing reaction conditions and steps recommended by the sequencing platform.

2>Set appropriate sequencing throughput and read length according to library concentration and sequencing depth requirements.

3. Sequencing monitoring and quality control:

1> Monitor the sequencing quality in real time during the sequencing process, including indicators such as signal strength, sequencing accuracy, and read length distribution.

2> Timely discover and solve problems that arise during the sequencing process, such as sequencer failure, reagent contamination, etc.

4. Data analysis

1. Data preprocessing:

1>Quality control and preprocessing of raw sequencing data, including removal of low-quality sequences, removal of adapter sequences and repetitive sequences, etc.

2. Sequence alignment and assembly:

1>Select an appropriate reference genome for sequence alignment to ensure the accuracy and reliability of the alignment results.

2> In the absence of a reference genome, you can try to use de novo assembly methods for genome construction.

3. Variation detection and annotation:

1>Use professional mutation detection tools to detect SNP, INDEL, CNV and other mutations.

2>Annotate and classify the detected variants and assess their potential clinical significance or biological function.

4. Results interpretation and reporting:

1>Interpret and analyze the results based on the experimental purpose and clinical background.

2>Write clear and accurate results reports, including experimental methods, data quality, mutation detection results and conclusions, etc.

5. Other operating skills

1. Experimental records and file management:

1>Record the experimental process, parameter settings, result data and other information in detail.

2>Establish a complete file management system to ensure the traceability and security of experimental data.

2. Training and technical support:

1>Carry out systematic training and assessment for experimental personnel to ensure that they master the operating skills of NGS workstations.

2> Provide necessary technical support and maintenance services to ensure the stable operation and efficient use of NGS workstations.

In the vast field of exploring and applying NGS workstations, mastering these operating skills is not only the key to successful experiments, but also an important cornerstone for promoting scientific research and technological innovation. As technology continues to advance and become more widely used, new operating methods and best practices will continue to emerge. Therefore, continuous learning, active communication and flexible application of these skills are essential for every NGS worker.