We are using a blunt-end ligation procedure to add barcoded, truncated adapter to the fragmented end-repaired DNA (Stiller et al. 2009 (link)). Specifically, one of the two truncated partially double-stranded adapters includes a 6-mer molecular barcode that is directly ligated to the blunted and 5′-phosphorylated DNA fragments and is therefore detected in the first six cycles of the first sequencing read. Because the adapters are not 5′-phosphorylated (to prevent adapter dimer formation and to reduce cost), a nick fill-in-step has to be performed before enrichment PCR, which then completes the truncated adapter sites so that the libraries can be sequenced (Fig. 2A). This PCR finishes the library preparation for the two WGS applications, but not for hybrid selection. For hybrid selection, we have modified the protocol so that the enrichment PCR (to complete the adapter to full length) is performed after hybrid capture, since we have found that the long adapters interfere with hybrid capture (see Supplemental Notes, “Influence of Adapter Length in Pooled Hybrid Capture”). No indexing read is needed to read out the internal barcode, but because cluster identification is performed in these cycles in the Illumina technology, care has to be taken to equally balance the four nucleotides at any of the six positions within the barcodes that will be sequenced together. Reaction conditions and overviews about the procedure can be found in Figures 1 and 2A (Supplemental Notes, “Sample Barcoding”) and Supplemental Figures S1, S3, and S4.