Multiple BioNano Genome Maps to Further Improve the Sequence Assembly of Aegilops tauschii Genome

The use of a BioNano genome (BNG) map to assist sequence assembly, specifically for super-scaffolding and pseudomolecule construction, is limited by gaps in the map. One of the attributes is those closely located nickase restriction site pairs on opposite DNA strands (fragile site pairs, FSPs), making such regions prone to breakage during DNA labeling and electrophoresis. The analysis of two BNG map assemblies for Aegilops tauschii acc. AL8/78 from independently collected Nt.BspQI nickase labelled molecules demonstrates a total of 1,148 gaps (out of 2,539 gaps calculated from the BNG map assembly with longer N50) were shared by both BNG maps on a whole-genome scale. Of these shared gaps, 74% were further confirmed by the prediction of FSPs from the sequence assembly of Ae. tauschii AL8/78, and over 98% of the distances within the FSPs are less than 200 bp. To bridge these “meant-to-be” breakages, we intended to use a second nickase, however, for Ae. tauschii genome, other nickases either introduced more FSPs that made molecules more fragile or were unstable. Thus alternatively, we utilized nickase site polymorphism (NSP) to alter the distribution of these fragile regions across different Ae. tauschii accessions. An additional BNG map was then constructed for Ae. tauschii acc. CIae23 using Nt.BspQI nickase as well. Our results show that NSP among the two Ae. tauschii genotypes was effective that the locations of 629 (out of confirmed 1,148) FSPs were altered and the use of the additional BNG map did improve the ordering and orienting of super-scaffolds on the pseudomolecules. This work is a part of the NSF-funded Project IOS-1238231 to generate a reference sequence for the genome of Ae. tauschii (http://aegilops.wheat.ucdavis.edu/ATGSP/).