Overview of the HD-Marker approach. (A) Probe preparation: Probes can be either column- or array-synthesized, with the latter allowing extremely high multiplex levels (up to 12,000 markers) at a very low probe cost (∼$0.001 per base). The diagram shows the preparation of single-stranded probes from an array-synthesized oligo pool through the steps of PCR amplification, enzyme digestion, and isolation by magnetic beads. (B) In-solution hybridization: Highly parallel probe hybridization is achieved in a single tube, with each probe consisting of a locus-specific portion and a universal PCR primer-binding portion. The gap between two probes covers the locus of interest (e.g., SNPs, microsatellites/SSRs or indels), which is filled in by a polymerase, followed by ligation of the extended LSP to the downstream LSP, creating a molecule that can be amplified by PCR. (C) Preparation of an HD-Marker library begins with attaching the genomic DNA to a solid support and then using two locus-specific probes (LSPs) to hybridize to the immobilized DNA. Through highly specific extension, ligation, and amplification steps, the libraries prepared from different samples can be pooled for high-throughput sequencing on a preferred NGS platform.
