Measuring clone size using the tag system. (A) The depiction above shows that shear site variations are not able to cover all sister cells in large clones. As the number of the sister cells in a given clone increases, the probability of DNA shearing at the same site increases. (B) Prior to PCR, we incorporated 8-bp random tags into each DNA fragment to uniquely mark them. Random tags could theoretically provide approximately 65,536 variations. The number of potential variations is expected to amply cover large numbers of the sister cells. (C) The tag information was used to remove PCR duplicates and to estimate the original number of starting fragments. If the fragments had the same shear sites but different tags, they were counted separately. For example, here five different combinations of tags and shear sites represent five infected cells. (D) Samples: S-1, S-2, S-3, and S-4 were analyzed by the final optimal condition (Bowtie parameters: -v 3 - - best, and filtering condition: (merging approach) JT-10). Clone size was measured by tags only or by the combination of shear sites and tags. The covered variations were (393,142, 1751, and 2675) and (269, 119, 1192, and 2038), respectively.