Analytic Validation of Optical Genome Mapping in Hematological Malignancies.

Structural variations (SVs) play a key role in the pathogenicity of hematological malignancies. Standard-of-care (SOC) methods such as karyotyping and fluorescence in situ hybridization (FISH), which have been employed globally for the past three decades, have significant limitations in terms of resolution and the number of recurrent aberrations that can be simultaneously assessed, respectively. Next-generation sequencing (NGS)-based technologies are now widely used to detect clinically significant sequence variants but are limited in their ability to accurately detect SVs. Optical genome mapping (OGM) is an emerging technology enabling the genome-wide detection of all classes of SVs at a significantly higher resolution than karyotyping and FISH. OGM requires neither cultured cells nor amplification of DNA, addressing the limitations of culture and amplification biases. This study reports the clinical validation of OGM as a laboratory-developed test (LDT) according to stringent regulatory (CAP/CLIA) guidelines for genome-wide SV detection in different hematological malignancies. In total, 60 cases with hematological malignancies (of various subtypes), 18 controls, and 2 cancer cell lines were used for this study. Ultra-high-molecular-weight DNA was extracted from the samples, fluorescently labeled, and run on the Bionano Saphyr system. A total of 215 datasets, Inc.luding replicates, were generated, and analyzed successfully. Sample data were then analyzed using either disease-specific or pan-cancer-specific BED files to prioritize calls that are known to be diagnostically or prognostically relevant. Sensitivity, specificity, and reproducibility were 100%, 100%, and 96%, respectively. Following the validation, 14 cases and 10 controls were run and analyzed using OGM at three outside laboratories showing reproducibility of 96.4%. OGM found more clinically relevant SVs compared to SOC testing due to its ability to detect all classes of SVs at higher resolution. The results of this validation study demonstrate the superiority of OGM over traditional SOC methods for the detection of SVs for the accurate diagnosis of various hematological malignancies.

A high-quality bonobo genome refines the analysis of hominid evolution.

The divergence of chimpanzee and bonobo provides one of the few examples of recent hominid speciation1,2. Here we describe a fully annotated, high-quality bonobo genome assembly, which was constructed without guidance from reference genomes by applying a multiplatform genomics approach. We generate a bonobo genome assembly in which more than 98% of genes are completely annotated and 99% of the gaps are closed, including the resolution of about half of the segmental duplications and almost all of the full-length mobile elements. We compare the bonobo genome to those of other great apes1,3-5 and identify more than 5,569 fixed structural variants that specifically distinguish the bonobo and chimpanzee lineages. We focus on genes that have been lost, changed in structure or expanded in the last few million years of bonobo evolution. We produce a high-resolution map of incomplete lineage sorting and estimate that around 5.1% of the human genome is genetically closer to chimpanzee or bonobo and that more than 36.5% of the genome shows incomplete lineage sorting if we consider a deeper phylogeny including gorilla and orangutan. We also show that 26% of the segments of incomplete lineage sorting between human and chimpanzee or human and bonobo are non-randomly distributed and that genes within these clustered segments show significant excess of amino acid replacement compared to the rest of the genome.

Haplotype-resolved genomes provide insights into structural variation and gene content in Angus and Brahman cattle.

Inbred animals were historically chosen for genome analysis to circumvent assembly issues caused by haplotype variation but this resulted in a composite of the two genomes. Here we report a haplotype-aware scaffolding and polishing pipeline which was used to create haplotype-resolved, chromosome-level genome assemblies of Angus (taurine) and Brahman (indicine) cattle subspecies from contigs generated by the trio binning method. These assemblies reveal structural and copy number variants that differentiate the subspecies and that variant detection is sensitive to the specific reference genome chosen. Six genes with immune related functions have additional copies in the indicine compared with taurine lineage and an indicus-specific extra copy of fatty acid desaturase is under positive selection. The haplotyped genomes also enable transcripts to be phased to detect allele-specific expression. This work exemplifies the value of haplotype-resolved genomes to better explore evolutionary and functional variations.

Author Correction: The axolotl genome and the evolution of key tissue formation regulators.

In the originally published version of this Article, the sequenced axolotl strain (the homozygous white mutant) was denoted as 'D/D' rather than 'd/d' in Fig. 1a and the accompanying legend, the main text and the Methods section. The original Article has been corrected online.

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Combination of short-read, long-read, and optical mapping assemblies reveals large-scale tandem repeat arrays with population genetic implications.

Accurate and contiguous genome assembly is key to a comprehensive understanding of the processes shaping genomic diversity and evolution. Yet, it is frequently constrained by constitutive heterochromatin, usually characterized by highly repetitive DNA. As a key feature of genome architecture associated with centromeric and subtelomeric regions, it locally influences meiotic recombination. In this study, we assess the impact of large tandem repeat arrays on the recombination rate landscape in an avian speciation model, the Eurasian crow. We assembled two high-quality genome references using single-molecule real-time sequencing (long-read assembly [LR]) and single-molecule optical maps (optical map assembly [OM]). A three-way comparison including the published short-read assembly (SR) constructed for the same individual allowed assessing assembly properties and pinpointing misassemblies. By combining information from all three assemblies, we characterized 36 previously unidentified large repetitive regions in the proximity of sequence assembly breakpoints, the majority of which contained complex arrays of a 14-kb satellite repeat or its 1.2-kb subunit. Using whole-genome population resequencing data, we estimated the population-scaled recombination rate (ρ) and found it to be significantly reduced in these regions. These findings are consistent with an effect of low recombination in regions adjacent to centromeric or subtelomeric heterochromatin and add to our understanding of the processes generating widespread heterogeneity in genetic diversity and differentiation along the genome. By combining three different technologies, our results highlight the importance of adding a layer of information on genome structure that is inaccessible to each approach independently.

Whole-genome mutational burden analysis of three pluripotency induction methods.

There is concern that the stresses of inducing pluripotency may lead to deleterious DNA mutations in induced pluripotent stem cell (iPSC) lines, which would compromise their use for cell therapies. Here we report comparative genomic analysis of nine isogenic iPSC lines generated using three reprogramming methods: integrating retroviral vectors, non-integrating Sendai virus and synthetic mRNAs. We used whole-genome sequencing and de novo genome mapping to identify single-nucleotide variants, insertions and deletions, and structural variants. Our results show a moderate number of variants in the iPSCs that were not evident in the parental fibroblasts, which may result from reprogramming. There were only small differences in the total numbers and types of variants among different reprogramming methods. Most importantly, a thorough genomic analysis showed that the variants were generally benign. We conclude that the process of reprogramming is unlikely to introduce variants that would make the cells inappropriate for therapy.

Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays.

Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.