One study suggests that some of the sequence variations found in DNA samples may actually be caused by damage during sample processing. Researchers from NEB (New England Biolabs) have developed a new method of evaluating these lesions and suggested DNA repair enzymes are used in the preparation of the sample to correct this problem.
"This damage is more common than we expected," says Thomas Evans, one of the authors of the article on Nature's February 16 issue. "This error is likely to confuse the real low-frequency somatic cells mutation.
We all know that DNA samples extracted from paraffin-embedded tissues are prone to breakage and chemical modification from old samples with long shelf life or formalin fixation, leading to mutations that do not exist in living organisms themselves. However, the latest research shows that, in fact, any DNA samples are likely to occur such artificial mutagenesis damage, such as DNA sonication, that is, the use of acoustic energy to stimulate the DNA fragments for amplification and sequencing, it is possible to induce mutation Of oxidative damage.
Such mutations sometimes occur only in a small number of samples and therefore do not cause much trouble. However, in cancer biology, more and more emphasis on subcloning recognition, as well as in the plasma detection of free tumor DNA mutation, the two in the sample ratio is very small. "This article is a good warning article," said Marc Ladanyi, a molecular oncologist at the Sloan / Kardlin Cancer Memorial Research Center in New York, who is not part of the study.
NEB researchers Laurence Ettwiller et al. Designed a new computational approach to calculate the extent of this damage in DNA sequencing samples. The basis of this algorithm is that oxidative damage to DNA during sonication translates guanine into 8-oxoguanine, which is used as a thymidine during sequencing.
Compared to the sequencing fragments of the two complementary strands, these altered guanines are thought to be mismatched, and one chain reads out thymine, but the complementary strand shows cytosine (which can be paired with guanine). On the other hand, the naturally occurring guanine-thymidine mutation will appear itself to paired adenine. Therefore, this method of calculation by comparing the first and second sequencing results, assess the degree of mismatch of thymine to determine the extent of damage.
Researchers used this method, named Global Imbalance Value (GIV, the overall imbalance value of the calculation method, the detection of thousands of human genome and cancer genome Atlas project data, the results found that thousands of human genome data in 41% Indicating the imbalance of injury scores, cancer genome Atlas project is up to 73%
The researchers suggested that the DNA repair enzyme mixture be added to the DNA sample during the preparation, resulting in a stable oxidative damage rate.
"(This article) presents a solution to the use of enzyme cocktail mixtures to repair DNA," Ladanyi said. But he also pointed out that "the author of the article from the NEB, the solution is to use the NEB repair system, so there is a conflict of interest."
In this regard, Ettwiller said that although the study group used NEB enzyme to repair damaged DNA samples, but they did not say that this will apply to all DNA preparation process.
"We did sell this enzyme mix for repairing upstream DNA, but we did not pack it, and we will continue to evaluate it," Evans said.