Baehr Stephan, Gout Jean-Francois, Reyes Lauren, Ray Haimanti, Lynch Michael
Center for Mechanisms of Evolution, Biodesign Institute, Arizona State University.
School of Life Sciences, Arizona State University.
bioRxiv. 2025 Jul 31:2025.07.30.667586. doi: 10.1101/2025.07.30.667586.
DNA mutation is the ultimate source of all heritable genetic and phenotypic variation. On human timescales, DNA mutation results in the evolution of antibiotic resistance, viral resistance to vaccines, the emergence human cancers, and more. Precise measurement of DNA mutation is therefore desirable to rapidly detect and analyze low frequency mutations in a population of cells. Precise measurement of DNA mutation by high throughput sequencing has been hindered by sequencing error rates, which occur at rates 1×10 to 1×10 per base sequenced. Previous work on circle sequencing had pushed a putative sequencing error rate down to roughly 2.8×10 per base for genomic yeast DNA in the absence of DNA repair enzymes, where the expected number is 4.7×10 per base per generation. Through revision of the assay, we are now capable of taking 125ng of genomic DNA and obtaining mutation rate estimates with a sequencing resolution floor of 2×10 per base in the absence of repair enzymes, a resolution improvement of over 3 orders of magnitude. In practice, circle-seq recovers some of the mutation spectrum of mismatch-repair deficient , although some signature of C → T and G → A errors remain present. Curiously, it calls a mutation rate of 2×10 per base per generation, lower than expected, underscoring the difficulty of directly comparing mutation rates per base to MA mutation rates per site per generation. This protocol readily recognizes Covaris ultrasonication as mutagenic in library preps, with a mutation spectrum dominated by G:C → A:T, G:C → C:G, and G:C → T:A errors. The protocol is either detecting DNA damage that has yet to be repaired, is significantly biased in its ability to detect mutations, or is not yet sensitive enough to detect mutation rates of MMR- cells. The protocol may have some use in measuring mutation abundance from tissue samples, and sources of DNA damage. The protocol also highlights some biases inherent to single-stranded DNA (ssDNA) cyclization, including a 10-bp periodicity which echoes the constraints of dsDNA cyclization.
DNA突变是所有可遗传的基因和表型变异的最终来源。在人类时间尺度上，DNA突变导致抗生素耐药性的演变、病毒对疫苗的耐药性、人类癌症的出现等等。因此，精确测量DNA突变对于快速检测和分析细胞群体中的低频突变是很有必要的。通过高通量测序精确测量DNA突变一直受到测序错误率的阻碍，测序错误率在每测序一个碱基时为1×10到1×10。先前关于环状测序的工作在没有DNA修复酶的情况下，已将基因组酵母DNA的假定测序错误率降至每碱基约2.8×10，而预期数量为每代每碱基4.7×10。通过对检测方法的改进，我们现在能够使用125ng的基因组DNA，并在没有修复酶的情况下获得测序分辨率下限为每碱基2×10的突变率估计值，分辨率提高了3个多数量级。实际上，环状测序恢复了错配修复缺陷的一些突变谱，尽管仍然存在一些C→T和G→A错误的特征。奇怪的是，它得出的每代每碱基突变率为2×10，低于预期，这凸显了直接将每碱基突变率与每代每位点的MA突变率进行比较的困难。该方案很容易识别Covaris超声处理在文库制备中具有诱变作用，其突变谱以G:C→A:T、G:C→C:G和G:C→T:A错误为主。该方案要么检测到尚未修复的DNA损伤，要么在检测突变的能力上存在显著偏差，要么对检测MMR - 细胞的突变率还不够敏感。该方案在测量组织样本中的突变丰度以及DNA损伤来源方面可能有一定用途。该方案还突出了单链DNA（ssDNA）环化固有的一些偏差，包括一个10碱基的周期性，这与双链DNA环化的限制相呼应。
