BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

Helena Staňková,Mingcheng Luo,Jan Vrána,Hana Šimková,Satomi Hayashi,Saki Chan,Jaroslav Doležel,Marie Kubaláková,Paul Visendi,Jacqueline Batley,Alex R Hastie,David Edwards,Zuzana Tulpová

doi:10.1111/pbi.12513

Abstract

SummaryThe assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC‐by‐BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high‐resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high‐resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome‐scale analysis of repetitive sequences and revealed a ~800‐kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone‐by‐clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC‐contig physical map and validate sequence assembly on a chromosome‐arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome‐by‐chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.

Highlights

Recent progress in understanding eukaryotic genome structure and function lead to the realization that a majority of genome sequences is transcribed and that, in addition to protein coding sequences, the so-called noncoding DNA may be functionally significant (ENCODE Project Consortium, 2012)
We chose the short arm of chromosome 7D (7DS) with the size of 381 Mb (Gill et al, 1991; Safar et al, 2010) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays to create a de novo genome map
The genome map of the 7DS chromosome arm of wheat was built from molecules treated by the nicking enzyme Nt.BspQI

Summary

Introduction

Recent progress in understanding eukaryotic genome structure and function lead to the realization that a majority of genome sequences is transcribed and that, in addition to protein coding sequences, the so-called noncoding DNA may be functionally significant (ENCODE Project Consortium, 2012). Unexpected plasticity of eukaryotic genomes, and functional significance of copy number and structural variation, has been revealed (Zarrei et al, 2015) These observations underline the need for high-quality reference genome sequences, which are a prerequisite to study these phenomena and discover genome features other than genes underlying traits of agronomic importance. The published reference genome sequences obtained using whole-genome shotgun strategies may suffer from extensive mis-assemblies and comprise gaps (Ganapathy et al, 2014; Pendleton et al, 2015; Ruperao et al, 2014). Wider application of technologies providing reads in the kilobase range, such as single molecule real-time sequencing adopted by Pacific Biosciences (Chaisson et al, 2015), and nanopore technologies (Mikheyev and Tin, 2014) promise to improve the quality

Results

Discussion

Conclusion