Simulated reads. Simulated reads are generated with mutations at each end of the amplicon insert. The minimum distance between the Uracil base in the primer and the 3’end of the primer is indicated by u. Only Thymine bases in the primer can be replaced by a Uracil base for primer digestion, so the actual distance from the Uracil base to the 3’ end of the primer (indicated by d) is dependent on the primer sequence, and can be much greater than u. The figure shows a scenario where d is the same as u on one end of the amplicon, but d is much greater than u on the other end of the amplicon.