previous run(s). Importantly, the resequenced sample must be a technical replicate; performing the protocol again on the same biological sample will not work. The SSCS:DCS ratio is the other key factor. A ratio of two is the theoretically ideal and would indicate that every SSCS can find its partner and form a DCS. We typically obtain SSCS:DCS ratios between four and ten, with an average of ~6. An excessively high ratio (i.e., >14–15) can occur for several reasons. If peak family sizes are too small, then families with at least three members might not form from both DNA strands. This issue is readily overcome by increasing the family size, as noted above, although it does not appreciably improve above peak sizes of 5–6 (Fig. 5b). If a poor ratio is seen despite adequate family sizes, this suggests that only one of the two DNA strands was successfully ligated and amplified during PCR. In such cases, the sample library will need to be remade in order to obtain more data, with attention to optimizing ligation conditions and library tailing steps.
