Fosmid-based physical mapping of the Histoplasma capsulatum genome

Abstract

A fosmid library representing 10-fold coverage of the Histoplasma capsulatum G217B genome was used to construct a restriction-based physical map. The data obtained from three restriction endonuclease fingerprints, generated from each clone using BamHI, HindIII, and PstI endonucleases, were combined and used in FPC for automatic and manual contig assembly builds. Concomitantly, a whole-genome shotgun (WGS) sequencing of paired-end reads from plasmids and fosmids were assembled with PCAP, providing a predicted genome size of up to 43.5 Mbp and 17% repetitive DNA. Fosmid paired-end sequences in the WGS assembly provide anchoring information to the physical map and result in joining of existing physical map contigs into 84 clusters containing 9551 fosmid clones. Here, we detail mapping the Histoplasma capsulatum genome comprehensively in fosmids, resulting in an efficient paradigm for de novo sequencing that uses a map-assisted whole genome shotgun approach.

Figure 1

Stacked fingerprints were generated by offsetting the mobility rates within each bands file. Here, we incorporate up to three fingerprints for each clone as the combined bands file, and the mobility rates of each HindIII, BamHI, and PstI fingerprint were changed to 1–4000, 4001–8000, and 8001–12000, respectively. During manual editing, FPC builds added bands file of clones missing either one fingerprint (blank lanes in the BamHI digest) or two fingerprints (blank lanes in both HindIII and BamHI digests).

Figure 2

MapLink provides anchoring information between the WGS assembly contigs (represented as dotted lines) and the physical map clusters (solid bars). Physical map cluster 2501, represented by 67 overlapping fosmid clones built in FPC, anchors to WGS assembly Supercontig Merge 57. This Supercontig is composed of three contigs, that is, 26, 22, and 19 with sizes of 107.8, 136.2, and 15.0 kb, respectively. Contigs 26 and 22 are anchored by gap-spanning fosmid clones L_AAZ090H07, L_AAZ088E04, and L_AAZ059B11 (solid lines), and contigs 22 and 19 are anchored by L_AAZ081F09 and L_AAZ089B12 (solid lines). In contig 26, L_AAZ036H02 (dashed line) anchors position 80,390 and links to Supercontig Merge 20. Contig 19 contains end sequence of L_AAZ091F01 and L_AAZ080C03 (dashed lines) and anchors map cluster 5901 at positions 460 and 7700 in Supercontig Merge 57. Map cluster 5901 also anchors Supercontig Merge 81, and this anchor provides long-range linking information between WGS assembly Supercontigs Merge 57 and Merge 81. This figure is not drawn to scale, and linkage information does not indicate sequence overlap.