Sequencing library construction for DS is similar to the standard Illumina library preparation protocol. The protocol follows the basic standard steps of DNA shearing by sonication, size selection, end repair, 3′ dA-tailing, adapter ligation, PCR amplification and, optionally, targeted DNA capture. We have made several important updates and optimizations to the protocol since its initial publication, which have substantially increased its reliability and reproducibility. Specifically, we have re-designed the sequencing adapters to allow for more efficient dA-tailing of the sample DNA. As part of this new design, the adapters require a different synthesis method that we have included in Boxes 1 and 2. An additional change in the protocol involves the amount of DNA used during the PCR step. In the original publication, we specified that ~40 attomoles of DNA was optimal. However, we have since determined that the PCR input amount depends on a number of factors, including the number of reads devoted to a sample, the use of targeted DNA capture, genome and target size, and gene and target copy number. We formally outline the relationship between these
