Operation process of NGS library construction workstation

The operation steps of NGS (Next Generation Sequencing) library construction workstation usually involve multiple complex molecular biology processes, aiming to convert DNA or RNA samples to be sequenced into libraries suitable for high-throughput sequencers. The following is an overview of the operating steps of the NGS library construction workstation based on general principles, but the specific steps may vary depending on different library construction methods, sample types, and equipment and reagents used. The content of this article is for reference only:

Overview of NGS library building workstation operation steps

1. Sample preparation

1> Obtain samples: Extract DNA or RNA from organisms (such as cells, tissues, blood, etc.).

2>Quality control: Evaluate the quality and quantity of samples to ensure that they meet the requirements for database construction.

2. Nucleic acid fragmentation

1>DNA fragmentation: For DNA library construction, physical methods (such as ultrasound, enzyme digestion) are usually used to break the DNA into small fragments of a certain length (such as 200-500bp).

2>RNA fragmentation: For RNA library construction, especially mRNA library construction, it is necessary to purify the mRNA first, and then carry out fragmentation processing (such as using metal ions, enzyme treatment, etc.).

3. End repair and tailing

1>DNA end repair: Use end repair enzymes to phosphorylate the fragmented DNA at the 5' end and add A to the 3' end to facilitate subsequent adapter ligation.

2>RNA reverse transcription: For RNA library construction, the fragmented RNA needs to be reverse transcribed into cDNA, and then subjected to DNA-like end repair and tailing processing.

4. Connector connection

1>Adapter design: Design appropriate adapter sequences according to the requirements of the sequencing platform.

2>Adapter ligation: Use DNA ligase to connect adapters to both ends of the processed DNA or cDNA fragments.

5. Library amplification

1>PCR amplification: Amplify the DNA or cDNA fragment connected to the adapter through PCR reaction to increase the concentration of the target sequence in the library.

2>Quality control: Perform quality control on the amplified library to evaluate its concentration, purity and fragment size distribution.

6. Library purification

1> Magnetic bead purification: Use magnetic beads and other methods to remove impurities (such as primers, salt ions, etc.) in the library and improve the purity of the library.

2>Quantitative detection: Use qPCR and other methods to quantitatively detect the purified library to facilitate sample distribution during subsequent sequencing.

7. Library quality inspection and storage

1>Quality inspection: Perform quality inspection on the final library to ensure that it meets the sequencing requirements.

2>Storage: Store qualified libraries under appropriate conditions (such as -20°C or -80°C) for subsequent sequencing.

8. Precautions

1>During the entire library construction process, experimental conditions (such as temperature, time, enzyme amount, etc.) need to be strictly controlled to ensure the accuracy and repeatability of experimental results.

2> Different library construction methods (such as traditional library construction, transposase library construction, amplicon library construction, etc.) will have different operating steps. The appropriate library construction method should be selected based on experimental needs.

3>When using the NGS library construction workstation, you should carefully read the equipment instructions and reagent operating instructions, and follow the laboratory's safety operating procedures.

As can be seen from the above operation process, the specific operation steps of the NGS library construction workstation are highly dependent on the equipment model, reagent type and experimental design used. Therefore, before actual implementation, consult the equipment supplier for professional advice or extensively consult the latest Scientific literature, for detailed and precise operational guidance, is crucial. Such preparatory work can ensure the smooth progress of the experimental process and maximize the accuracy and reliability of experimental results.