Sequencing Key Terms

Q:What is second-generation sequencing?

A:Second-generation sequencing is an advanced genetic testing method. Compared with traditional genetic testing methods, such as PCR, microarray method, it has a large throughput (throughput means that the number of samples detected at the same time, simultaneous detection of more genes, so it is also known as high-throughput sequencing), detection of multiple types of genetic variation (can be detected at the same time mutation, fusion, copy number variations, etc.). Compared with the first generation sequencing, it is more sensitive and can detect more types of genes.

Q: Why is second-generation sequencing an algorithm-based test?

A:. Second-generation sequencing is mainly divided into the detection process and the two major stages of the letter analysis, after the machine, but also through the automatic data analysis and manual review of the letter analysis, and the letter analysis, can be understood as the results of the test according to a specific algorithm to reasoning about the genetic variability.

Below is the process standard of the biometric analysis, which can be understood.

Q: How to evaluate the accuracy of sequencing results?

A: On the one hand, it is the quality management system of the testing laboratory itself, and on the other hand, it is the inter-room quality evaluation of the high-throughput sequencing testing process and BioSignal analysis organized by national authoritative institutions, such as the Commission on Health, that is, the quality assessment activities organized by the state to organize the examination, the state assessment scoring, and the release of the results. Clinical laboratories that can achieve perfect scores in the two quality assessment activities of the detection process and bioconfidence analysis indicate that the sequencing quality is reliable.

Q: What does sequencing sensitivity mean?

A: Sensitivity refers to the ability to find one variant allele out of a number of alleles. Tissue and blood require different sensitivities, one percent for tissue and one part per thousand for blood.

Q: Is the more sensitive the better?

A: From the clinical point of view, sensitivity and specificity cannot be optimized at the same time. On the one hand, the more sensitive it is, the rarer the proportion of mutated cells in the tumor cells is, and if the drug is used accordingly, the result is likely to be inconsistent with the actual clinical situation. On the other hand, too much sensitivity may sacrifice specificity, i.e., the possibility of false positives. Therefore, the most clinically realistic sensitivity is that one percent for tissue and one part per thousand for blood is sufficient.

Q:What is sequencing depth?

A: Sequencing depth refers to the number of times the same locus has been scanned and detected; the larger the value, the deeper the depth. Sequencing depth is not sensitivity, but it is related to sensitivity. Sequencing depth is divided into depth before de-duplication and depth after de-duplication. Duplication means repetition because the sample DNA will have an amplification process, which means that there is a lot of DNA that is repetitive and redundant and needs to be removed. The required sequencing depth for tissue is 1000% before de-weighting and 500% after de-weighting. The required sequencing depth for blood is 10,000 into before de-redundancy and 5,000 into after removal.

Q: What is cfDNA and what is ctDNA?

A: cfDNA is the free DNA in the blood (also pleural fluid, cerebral effusion, etc.), also known as circulating cell-free DNA, and there are a lot of cells that can release cfDNA, which comes from many sources, and is very complex.

ctDNA specifically refers to circulating tumor DNA, which is DNA present in blood, pleural fluid, and released from tumor cells, and is a very small fraction of cfDNA. The biggest difficulty in detecting ctDNA is to remove the background interference, that is, the vast majority of non-tumor cells released cfDNA interference. This makes the detection process and raw letter analysis more demanding.

Q: How do I answer the question of false positives and false negatives asked by my doctor?

A:Doctors who ask such questions are essentially concerned about the quality of the test. Therefore, the reliability of the test can be confirmed by a double perfect score in the quality assessment of the wet test (detection process, with wet reagents involved) and the dry test (raw letter analysis, without reagents involved).

In addition, low-frequency mutations are reconfirmed by microarray technology, which minimizes the problem of false positives and false negatives.

In addition, blood cfDNA testing, using the UMI labeling method, is also a way to avoid false positives and false negatives.