Whole-Genome Restriction Mapping by "Subhaploid"-Based RAD Sequencing: An Efficient and Flexible Approach for Physical Mapping and Genome Scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based "" linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of "subhaploid" fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6-14 kb), with up to 15-fold improvement of N50 (∼816 kb-3.7 Mb) and high scaffolding accuracy (98.1-98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies.

Dou J ，Dou H ，Mu C ，Zhang L ，Li Y ，Wang J ，Li T ，Li Y ，Hu X ，Wang S ，Bao Z ... -  《-》

MaGuS: a tool for quality assessment and scaffolding of genome assemblies with Whole Genome Profiling™ Data.

Scaffolding is an essential step in the genome assembly process. Current methods based on large fragment paired-end reads or long reads allow an increase in contiguity but often lack consistency in repetitive regions, resulting in fragmented assemblies. Here, we describe a novel tool to link assemblies to a genome map to aid complex genome reconstruction by detecting assembly errors and allowing scaffold ordering and anchoring. We present MaGuS (map-guided scaffolding), a modular tool that uses a draft genome assembly, a Whole Genome Profiling™ (WGP) map, and high-throughput paired-end sequencing data to estimate the quality and to enhance the contiguity of an assembly. We generated several assemblies of the Arabidopsis genome using different scaffolding programs and applied MaGuS to select the best assembly using quality metrics. Then, we used MaGuS to perform map-guided scaffolding to increase contiguity by creating new scaffold links in low-covered and highly repetitive regions where other commonly used scaffolding methods lack consistency. MaGuS is a powerful reference-free evaluator of assembly quality and a WGP map-guided scaffolder that is freely available at https://github.com/institut-de-genomique/MaGuS. Its use can be extended to other high-throughput sequencing data (e.g., long-read data) and also to other map data (e.g., genetic maps) to improve the quality and the contiguity of large and complex genome assemblies.

Madoui MA ，Dossat C ，d'Agata L ，van Oeveren J ，van der Vossen E ，Aury JM ... -  《BMC BIOINFORMATICS》

Improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data.

Long-read sequencing can overcome the weaknesses of short reads in the assembly of eukaryotic genomes; however, at present additional scaffolding is needed to achieve chromosome-level assemblies. We generated Pacific Biosciences (PacBio) long-read data of the genomes of three relatives of the model plant and assembled all three genomes into only a few hundred contigs. To improve the contiguities of these assemblies, we generated BioNano Genomics optical mapping and Dovetail Genomics chromosome conformation capture data for genome scaffolding. Despite their technical differences, optical mapping and chromosome conformation capture performed similarly and doubled N50 values. After improving both integration methods, assembly contiguity reached chromosome-arm-levels. We rigorously assessed the quality of contigs and scaffolds using Illumina mate-pair libraries and genetic map information. This showed that PacBio assemblies have high sequence accuracy but can contain several misassemblies, which join unlinked regions of the genome. Most, but not all, of these misjoints were removed during the integration of the optical mapping and chromosome conformation capture data. Even though none of the centromeres were fully assembled, the scaffolds revealed large parts of some centromeric regions, even including some of the heterochromatic regions, which are not present in gold standard reference sequences.

Jiao WB ，Accinelli GG ，Hartwig B ，Kiefer C ，Baker D ，Severing E ，Willing EM ，Piednoel M ，Woetzel S ，Madrid-Herrero E ，Huettel B ，Hümann U ，Reinhard R ，Koch MA ，Swan D ，Clavijo B ，Coupland G ，Schneeberger K ... -  《-》

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

The 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.

Staňková H ，Hastie AR ，Chan S ，Vrána J ，Tulpová Z ，Kubaláková M ，Visendi P ，Hayashi S ，Luo M ，Batley J ，Edwards D ，Doležel J ，Šimková H ... -  《-》

Improving Nelumbo nucifera genome assemblies using high-resolution genetic maps and BioNano genome mapping reveals ancient chromosome rearrangements.

Genetic and physical maps are powerful tools to anchor fragmented draft genome assemblies generated from next-generation sequencing. Currently, two draft assemblies of Nelumbo nucifera, the genomes of 'China Antique' and 'Chinese Tai-zi', have been released. However, there is presently no information on how the sequences are assembled into chromosomes in N. nucifera. The lack of physical maps and inadequate resolution of available genetic maps hindered the assembly of N. nucifera chromosomes. Here, a linkage map of N. nucifera containing 2371 bin markers [217 577 single nucleotide polymorphisms (SNPs)] was constructed using restriction-site associated DNA sequencing data of 181 F individuals and validated by adding 197 simple sequence repeat (SSR) markers. Additionally, a BioNano optical map covering 86.20% of the 'Chinese Tai-zi' genome was constructed. The draft assembly of 'Chinese Tai-zi' was improved based on the BioNano optical map, showing an increase of the scaffold N50 from 0.989 to 1.48 Mb. Using a combination of multiple maps, 97.9% of the scaffolds in the 'Chinese Tai-zi' draft assembly and 97.6% of the scaffolds in the 'China Antique' draft assembly were anchored into pseudo-chromosomes, and the centromere regions along the pseudo-chromosomes were identified. An evolutionary scenario was proposed to reach the modern N. nucifera karyotype from the seven ancestral eudicot chromosomes. The present study provides the highest-resolution linkage map, the optical map and chromosome level genome assemblies for N. nucifera, which are valuable for the breeding and cultivation of N. nucifera and future studies of comparative and evolutionary genomics in angiosperms.

Gui S ，Peng J ，Wang X ，Wu Z ，Cao R ，Salse J ，Zhang H ，Zhu Z ，Xia Q ，Quan Z ，Shu L ，Ke W ，Ding Y ... -  《-》