Next Generation Sequencing（NGS）Application Science

Application Science

High-throughput sequencing technology is widely used in tumor genetic testing. On the one hand, gene sequencing can identify the mutated genes of cancer and thus rapidly determine the targeted drugs; on the other hand, the sample type can be the patient's blood or even saliva, eliminating the need for traditional pathological sections and reducing the damage to the patient's organism during the diagnostic process. Here we will briefly introduce the process of NGS in the application of genetic testing of tumor samples.

1、Sample reception and quality control

Plasma sample requirements: the blood collection volume for ctDNA testing must be at least 8mL and transported to the central laboratory using the professional BunnyTube cfDNA Preservation Tube stored at room temperature. Tumor paraffin sample (FFPE) requirements:

① whole FFPE tissue;

② tissue size >0.5*0.5cm, cut out 5-10 sheets of unstained paraffin tissue, thickness 2-5μm;

③ tissue size <0.5*0.5cm, 10-15 sheets required, made into sections or directly stored into 1.5mL EP tube with tumor cell content not less than 10% and necrotic tissue not more than 70%.

The slide box should be marked with name, date of birth and date of collection, and stored at room temperature.

2、Sample DNA extraction and fragmentation

Different extraction kits are used for different sample types to extract the required DNA, and the DNA of large molecular fragments is broken into short sequence fragments of a certain length by ultrasonic fragmentation.

3、Library preparation and library enrichment

Library preparation: Prepared connectors are added to the extracted DNA fragments, and then the DNA selection and purification kits are used to remove large and small DNA fragments that do not meet the requirements, as well as excess connectors and other impurities. Library enrichment: Target DNA fragments are selected by adding blocking oligonucleotides - molecular hybridization (adding probes to be captured by hybridization with the target) - adsorption of probes (adding magnetic beads and thus adsorbing probes) - magnetic sedimentation (capturing target fragments with magnets and eluting off-target fragments).

4、Sequencing

5、Bioinformatics analysis and report generation

After the sample data is offline, the computing center analyzes the data using different analysis and computational methods according to the requirements of different experimental programs. A specific report is then generated based on the results of the bioinformatics analysis for physician interpretation.

BunnyTeeth Technology products are developed and designed specifically for NGS and are widely used in oncogene testing, NIPT (non-invasive prenatal screening) and macro-genome sequencing.