Figure 4
Adaptation of Concat-Seq to an alternative target enrichment workflow. (a) Schematic of Roche Nimblegen’s SeqCap workflow and its adaptation to ConcatSeq. Short DNA fragments, for example cell-free DNA, are prepared for adapter ligation by end-repair and A-tailing (ERAT). During adapter ligation, two types of ConcatSeq adapters are used (blue and yellow portions) instead of the Y-shaped adapters normally used in this step. The resulting library pool is then used for hybridization to biotinylated (green dots) hybrid capture probes (gray bars), and enriched targets are subsequently amplified in a post-capture PCR. The amplified material is then concatenated and processed as described in Fig. 1a. (b) Bioanalyzer DNA7500 gel image showing ladder [L], the non-concatenated amplicon pool (a pool of 4 amplicons from the EGFR locus) [N], the non-concatenated amplicon pool with adapters [A], and the concatenated sample [C]. As expected a shift of about 60 bp was observed between lanes [N] and [A] indicating successful ligation of ConcatSeq adapters to the original pool of amplicons. Banding pattern in lane [C] indicates depletion of monomers and accumulation of n-mers of higher degree. (c) Histogram depicting frequencies of fragment lengths after deconcatenation of EGFR-concatemer reads. The size of the vast majority of fragments coincides with the expected EGFR amplicon length (220 bp).
