Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula

Abstract

Background: Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner.

Results: Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly.

Conclusions: Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

Keywords: BioNano; Dovetail; Genome assembly; Medicago truncatula; Next generation sequencing; PacBio.

Fig. 1

Synteny alignment of partial chromosomes 4 and 8 between A17 and R108 confirms rearrangement of the long arms of the chromosomes

Fig. 2

Synteny alignment of partial A17 chromosomes 4 and 8 against syntenic regions in the R108 Illumina-based assembly (top panel), PacBio-based assembly (Pb, middle panel) as well as the gap-filled PbDtBn (v1.0) assembly (bottom panel)

Fig. 3

Schematic of the rearrangement between chromosomes 4 and 8 in A17 (left) compared to R108 (right). Green segments indicate homology to A17’s chromosome 4 while blue segments indicate homology to A17 chromosome 8. Red segments indicate sequences not present in the A17 reference). Breakpoint 1 (br1) is pinpointed to a 104 bp region (chr4:39,021,788-39,021,891) and includes a 100 bp gap. Breakpoint 2 (br2) is pinpointed to a 7665 bp region (chr8:33,996,308-34,003,972) and includes a 7663 bp gap. Breakpoint 3 (br3) is pinpointed to a 708 bp region (chr8: 34,107,285-34,107,992) and includes a 100 bp gap. Breakpoint 4 is pinpointed to a 277 bp region (chr8:34,275,249-34,275,525) and includes a 100 bp gap)