Abstract
BackgroundThe assembly of the bread wheat genome sequence is challenging due to allohexaploidy and extreme repeat content (>80%). Isolation of single chromosome arms by flow sorting can be used to overcome the polyploidy problem, but the repeat content cause extreme assembly fragmentation even at a single chromosome level. Long jump paired sequencing data (mate pairs) can help reduce assembly fragmentation by joining multiple contigs into single scaffolds. The aim of this work was to assess how mate pair data generated from multiple displacement amplified DNA of flow-sorted chromosomes affect assembly fragmentation of shotgun assemblies of the wheat chromosomes.ResultsThree mate pair (MP) libraries (2 Kb, 3 Kb, and 5 Kb) were sequenced to a total coverage of 89x and 64x for the short and long arm of chromosome 7B, respectively. Scaffolding using SSPACE improved the 7B assembly contiguity and decreased gene space fragmentation, but the degree of improvement was greatly affected by scaffolding stringency applied. At the lowest stringency the assembly N50 increased by ~7 fold, while at the highest stringency N50 was only increased by ~1.5 fold. Furthermore, a strong positive correlation between estimated scaffold reliability and scaffold assembly stringency was observed. A 7BS scaffold assembly with reduced MP coverage proved that assembly contiguity was affected only to a small degree down to ~50% of the original coverage.ConclusionThe effect of MP data integration into pair end shotgun assemblies of wheat chromosome was moderate; possibly due to poor contig assembly contiguity, the extreme repeat content of wheat, and the use of amplified chromosomal DNA for MP library construction.
Highlights
The assembly of the bread wheat genome sequence is challenging due to allohexaploidy and extreme repeat content (>80%)
Based on the BWA results we identified the number of mate pair (MP) reads aligning to contigs, the proportion of MP read pairs mapping to the same or different contigs, and the orientation of the MP reads that mapped to the same contig
Our results show that integration of MP data in shotgun assemblies of flow-sorted wheat chromosomes improves assembly contiguity and decrease gene space fragmentation, but that the degree of assembly statistics improvement is greatly affected by scaffolding stringency (Table 1, Table 3)
Summary
The assembly of the bread wheat genome sequence is challenging due to allohexaploidy and extreme repeat content (>80%). The aim of this work was to assess how mate pair data generated from multiple displacement amplified DNA of flow-sorted chromosomes affect assembly fragmentation of shotgun assemblies of the wheat chromosomes. The hexaploid wheat genome can be dissected to small parts by flow cytometric sorting of single chromosomes and chromosome arms [12,13]. This technological breakthrough has enabled production of wheat chromosome specific BAC-libraries [14] and facilitated construction of physical maps of hexaploid wheat chromosomes [15]. MDA is known to give rise to chimeras, which can bring down the utility of the amplified DNA [18]
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