Sequence-based physical mapping of complex genomes by whole genome profiling

Abstract

We present whole genome profiling (WGP), a novel next-generation sequencing-based physical mapping technology for construction of bacterial artificial chromosome (BAC) contigs of complex genomes, using Arabidopsis thaliana as an example. WGP leverages short read sequences derived from restriction fragments of two-dimensionally pooled BAC clones to generate sequence tags. These sequence tags are assigned to individual BAC clones, followed by assembly of BAC contigs based on shared regions containing identical sequence tags. Following in silico analysis of WGP sequence tags and simulation of a map of Arabidopsis chromosome 4 and maize, a WGP map of Arabidopsis thaliana ecotype Columbia was constructed de novo using a six-genome equivalent BAC library. Validation of the WGP map using the Columbia reference sequence confirmed that 350 BAC contigs (98%) were assembled correctly, spanning 97% of the 102-Mb calculated genome coverage. We demonstrate that WGP maps can also be generated for more complex plant genomes and will serve as excellent scaffolds to anchor genetic linkage maps and integrate whole genome sequence data.

Figure 1.

Overview of whole genome profiling technology. (A) BAC library: BAC clones are available in a 384-wells plate format. (B) BAC pooling and DNA extraction: DNA is extracted after pooling each row (24 BACs) and each column (16 BACs). (C) Template preparation and sequencing: Pooled BAC DNA is digested (EcoRI/MseI) and amplified after barcoded adapters are ligated for pool identification after sequencing on the Illumina GA platform. (D) Deconvolution: Sequence tags (30–50 per BAC) are assigned to individual BACs based on presence in one row and one column pool. (E) Contiging: Overlapping (sets of) sequence tags from individual BAC clones generate contigs. Together, these contigs represent a sequence-based physical map of the genome.

Figure 2.

Distribution of the number of tags per BAC, specified for all 4599 deconvoluted BACs and for the subset of 551 BACs that were not assembled in contigs (singleton BACs).

Figure 3.

Overview (A) and zoomed-in detail (B) of a typical BAC contig located on Arabidopsis chromosome 3. The sequence of the WGP tags, the chromosome number, and the base pair position in the chromosome of the first base of the WGP tag are shown. In each color-coded BAC, the presence of a WGP tag is indicated by “x.” Gaps in BACs represent missing tags due to insufficient deconvoluted reads.