Combinations of libraries with different insert sizes improve contig scaffolding. (a) All library data sets were normalized to 8.5× non-clonal physical genome coverage resulting in the use of approximately 130 million pairs for the PE library to several million pairs for the MPs. The scaffold N50 (y-axis) as determined by SSPACE is plotted against the total number of scaffolds (x-axis) for each individual library and for all combinations of libraries. Scaffolding results for the current genome reference (RGSC 3.4) are displayed as well. (b) Representative examples of the genomic loci on rat chromosome 18 that show major discordance between optical map and the RGSC 3.4 reference genome. MP-assisted scaffolding restored concordance between sequence scaffolds and optical maps. The top panel (black) represents the reference genome assembly with the vertical lines indicating predicted SwaI sites; the middle panel (red) represents optical map data obtained using SwaI digests; the lower panel represents the rescaffolded genome using the MP data. The indicated positions on chromosome 18 are according to the current RGSC 3.4 assembly. A large region of approximately 75 kb (top panel) that shows low concordance with the predicted path of the optical map (0.065 Mb–0.14 Mb), increased significantly after MP-scaffolding. The bottom panel shows another example of increased resemblance to optical mapping data (3.85 Mb–3.90 Mb). Order and placement of contigs was shifted in the new scaffold resulting in SwaI sites identical to the optical map.