Variant (COSM214499) is always high in 0% in NGS QC results, why?
Conclusion: the frequency of the deletion detected using NGS without UMI was inaccuracy.
Reason: this deletion happened in an ATM's area with 10 consecutive Ts, where makes the accuracy detection very difficult.
Firstly, the errors like "Stutters" can be introduced thought very PCR, including the ones in both the LP process and Sequencing process. Secondly, the "errors" in our detection system cannot be rectified because of lacking of UMIs. So the "errors" finally were detected as variants mistakenly.
In Chinese 结论:这个位点的VAF 在目前的建库条件下(即没有UMI情况下)NGS检测的结果不准确。
原因:这个突变为1个T的缺失,它发生在ATM基因一个连续10个T的区域,因此它的正确检测会非常困难。
一方面,PCR过程很容易在这一区域带来错误如复制滑脱,无论是建库过程的PCR还是测序过程的PCR。而另一方面,由于我们建库加入的接头没有分子标签(UMI),无法修正这些错误,最终导致这些PCR错误被“误检”为突变。
我虽然认同这个区域很容易产生PCR错误,但我还有一点concern, 如下:
我们的WT也是通过PCR 扩增的,所以后期有UMI之后,纠正后的结果还是一样的 - 我的意思是这个错误是我们制作WT过程中引入的,如果是这样,那我们就需要非常注意这样类型的区域(连续碱基区域或者STR区域)。
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