Long-Read Structural and Epigenetic Profiling of a Kidney Tumor-Matched Sample with Nanopore Sequencing and Optical Genome Mapping.

Carcinogenesis often involves significant alterations in the cancer genome architecture, marked by large structural and copy number variations (SVs and CNVs) that are difficult to capture with short-read sequencing. Traditionally, cytogenetic techniques are applied to detect such aberrations, but they are limited in resolution and do not cover features smaller than several hundred kilobases. Optical genome mapping and nanopore sequencing are attractive technologies that bridge this resolution gap and offer enhanced performance for cytogenetic applications. These methods profile native, individual DNA molecules, thus capturing epigenetic information. We applied both techniques to characterize a clear cell renal cell carcinoma (ccRCC) tumor's structural and copy number landscape, highlighting the relative strengths of each method in the context of variant size and average read length. Additionally, we assessed their utility for methylome and hydroxymethylome profiling, emphasizing differences in epigenetic analysis applicability.

Flow Sorting-Assisted Optical Mapping.

Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.

Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays.

Proteins and enzymes in the cell nucleus require physical access to their DNA target sites in order to perform genomic tasks such as gene activation and transcription. Hence, chromatin accessibility is a central regulator of gene expression, and its genomic profile holds essential information on the cell type and state. We utilized the E. coli Dam methyltransferase in combination with a fluorescent cofactor analogue to generate fluorescent tags in accessible DNA regions within the cell nucleus. The accessible portions of the genome are then detected by single-molecule optical genome mapping in nanochannel arrays. This method allowed us to characterize long-range structural variations and their associated chromatin structure. We show the ability to create whole-genome, allele-specific chromatin accessibility maps composed of long DNA molecules extended in silicon nanochannels.

Prospects of telomere-to-telomere assembly in barley: Analysis of sequence gaps in the MorexV3 reference genome.

The first gapless, telomere-to-telomere (T2T) sequence assemblies of plant chromosomes were reported recently. However, sequence assemblies of most plant genomes remain fragmented. Only recent breakthroughs in accurate long-read sequencing have made it possible to achieve highly contiguous sequence assemblies with a few tens of contigs per chromosome, that is a number small enough to allow for a systematic inquiry into the causes of the remaining sequence gaps and the approaches and resources needed to close them. Here, we analyse sequence gaps in the current reference genome sequence of barley cv. Morex (MorexV3). Optical map and sequence raw data, complemented by ChIP-seq data for centromeric histone variant CENH3, were used to estimate the abundance of centromeric, ribosomal DNA, and subtelomeric repeats in the barley genome. These estimates were compared with copy numbers in the MorexV3 pseudomolecule sequence. We found that almost all centromeric sequences and 45S ribosomal DNA repeat arrays were absent from the MorexV3 pseudomolecules and that the majority of sequence gaps can be attributed to assembly breakdown in long stretches of satellite repeats. However, missing sequences cannot fully account for the difference between assembly size and flow cytometric genome size estimates. We discuss the prospects of gap closure with ultra-long sequence reads.

Fine structure and transcription dynamics of bread wheat ribosomal DNA loci deciphered by a multi-omics approach.

Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head-to-tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and gene copy number, sequence composition, and expression status of particular arrays remain elusive, especially in complex genomes harboring multiple loci. Here we conducted a multi-omics study to decipher the 45S rDNA loci in hexaploid bread wheat. Coupling chromosomal genomics with optical mapping, we reconstructed individual rDNA arrays, enabling locus-specific analyses of transcription activity and methylation status from RNA- and bisulfite-sequencing data. We estimated a total of 6,650 rDNA units in the bread wheat genome, with approximately 2,321, 3,910, 253, and 50 gene copies located in short arms of chromosomes 1B, 6B, 5D, and 1A, respectively. Only 1B and 6B loci contributed substantially to rRNA transcription at a roughly 2:1 ratio. The ratio varied among five tissues analyzed (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG context in the silenced 5D locus as compared with the active 1B and 6B loci. In conclusion, a fine genomic organization and tissue-specific expression of rDNA loci were deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of wheat evolution and transcription regulation.

Long-read sequence assembly: a technical evaluation in barley.

Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

Accessing a Russian Wheat Aphid Resistance Gene in Bread Wheat by Long-Read Technologies.

Russian wheat aphid (RWA) ( Kurdjumov) is a serious invasive pest of small-grain cereals and many grass species. An efficient strategy to defy aphid attacks is to identify sources of natural resistance and transfer resistance genes into susceptible crop cultivars. Revealing the genes helps understand plant defense mechanisms and engineer plants with durable resistance to the pest. To date, more than 15 RWA resistance genes have been identified in wheat ( L.) but none of them has been cloned. Previously, we genetically mapped the RWA resistance gene into an interval of 0.83 cM on the short arm of chromosome 7D and spanned it with five bacterial artificial chromosome (BAC) clones. Here, we used a targeted strategy combining traditional approaches toward gene cloning (genetic mapping and sequencing of BAC clones) with novel technologies, including optical mapping and long-read nanopore sequencing. The latter, with reads spanning the entire length of a BAC insert, enabled us to assemble the whole region, a task that was not achievable with short reads. Long-read optical mapping validated the DNA sequence in the interval and revealed a difference in the locus organization between resistant and susceptible genotypes. The complete and accurate sequence of the region facilitated the identification of new markers and precise annotation of the interval, revealing six high-confidence genes. Identification of as the most likely candidate opens an avenue for its validation through functional genomics approaches.

The Dark Matter of Large Cereal Genomes: Long Tandem Repeats.

Reference genomes of important cereals, including barley, emmer wheat and bread wheat, were released recently. Their comparison with genome size estimates obtained by flow cytometry indicated that the assemblies represent not more than 88-98% of the complete genome. This work is aimed at identifying the missing parts in two cereal genomes and proposing techniques to make the assemblies more complete. We focused on tandemly organised repetitive sequences, known to be underrepresented in genome assemblies generated from short-read sequence data. Our study found arrays of three tandem repeats with unit sizes of 1242 to 2726 bp present in the bread wheat reference genome generated from short reads. However, this and another wheat genome assembly employing long PacBio reads failed in integrating correctly the 2726-bp repeat in the pseudomolecule context. This suggests that tandem repeats of this size, frequently incorporated in unassigned scaffolds, may contribute to shrinking of pseudomolecules without reducing size of the entire assembly. We demonstrate how this missing information may be added to the pseudomolecules with the aid of nanopore sequencing of individual BAC clones and optical mapping. Using the latter technique, we identified and localised a 470-kb long array of 45S ribosomal DNA absent from the reference genome of barley.

Integrated physical map of bread wheat chromosome arm 7DS to facilitate gene cloning and comparative studies.

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere.

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.

Chromosomal genomics facilitates fine mapping of a Russian wheat aphid resistance gene.

KEY MESSAGE: Making use of wheat chromosomal resources, we developed 11 gene-associated markers for the region of interest, which allowed reducing gene interval and spanning it by four BAC clones. Positional gene cloning and targeted marker development in bread wheat are hampered by high complexity and polyploidy of its nuclear genome. Aiming to clone a Russian wheat aphid resistance gene Dn2401 located on wheat chromosome arm 7DS, we have developed a strategy overcoming problems due to polyploidy and enabling efficient development of gene-associated markers from the region of interest. We employed information gathered by GenomeZipper, a synteny-based tool combining sequence data of rice, Brachypodium, sorghum and barley, and took advantage of a high-density linkage map of Aegilops tauschii. To ensure genome- and locus-specificity of markers, we made use of survey sequence assemblies of isolated wheat chromosomes 7A, 7B and 7D. Despite the low level of polymorphism of the wheat D subgenome, our approach allowed us to add in an efficient and cost-effective manner 11 new gene-associated markers in the Dn2401 region and narrow down the target interval to 0.83 cM. Screening 7DS-specific BAC library with the flanking markers revealed a contig of four BAC clones that span the Dn2401 region in wheat cultivar 'Chinese Spring'. With the availability of sequence assemblies and GenomeZippers for each of the wheat chromosome arms, the proposed strategy can be applied for focused marker development in any region of the wheat genome.