Application of full-genome analysis to diagnose rare monogenic disorders

Current genetic tests for rare diseases provide a diagnosis in only a modest proportion of cases. The Full-Genome Analysis method, FGA, combines long-range assembly and whole-genome sequencing to detect small variants, structural variants with breakpoint resolution, and phasing. We built a variant prioritization pipeline and tested FGA’s utility for diagnosis of rare diseases in a clinical setting. FGA identified structural variants and small variants with an overall diagnostic yield of 40% (20 of 50 cases) and 35% in exome-negative cases (8 of 23 cases), 4 of these were structural variants. FGA detected and mapped structural variants that are missed by short reads, including non-coding duplication, and phased variants across long distances of more than 180 kb. With the prioritization algorithm, longer DNA technologies could replace multiple tests for monogenic disorders and expand the range of variants detected. Our study suggests that genomes produced from technologies like FGA can improve variant detection and provide higher resolution genome maps for future application.

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

ABSTRACTDNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and among the most significant epigenetic modifications. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in the CpG context allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. For this method, a CpG methyltransferase was used to transfer an azide to cytosines from a synthetic S-adenosyl-l-methionine cofactor analog. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased two-fold by the addition of a nucleosidase that degrades the inactive by-product of the azide-cofactor after labeling, and prevents its inhibitory effect. We first used the method to determine the decline in global DNA methylation in chronic lymphocytic leukemia patients and then performed whole genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published methylation maps produced by bisulfite sequencing. Although mapping resolution is limited by optical detection to 500-1000 base pairs, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

Cenote-Taker 2 Democratizes Virus Discovery And Sequence Annotation

Viruses, despite their great abundance and significance in biological systems, remain largely mysterious. Indeed, the vast majority of the perhaps hundreds of millions of viral species on the planet remain undiscovered. Additionally, many viruses deposited in central databases like GenBank and RefSeq are littered with genes annotated as “hypothetical protein” or the equivalent. Cenote-Taker 2, a virus discovery and annotation tool available on command line and with a graphical user interface with free high-performance computation access, utilizes highly sensitive models of hallmark virus genes to discover familiar or divergent viral sequences from user-input contigs. Additionally, Cenote-Taker 2 uses a flexible set of modules to automatically annotate the sequence features of contigs, providing more gene information than comparable tools. The outputs include readable and interactive genome maps, virome summary tables, and files that can be directly submitted to GenBank. We expect Cenote-Taker 2 to facilitate virus discovery, annotation, and expansion of the known virome.

Application of Full Genome Analysis to Diagnose Rare Monogenic Disorders

Current genetic tests for rare diseases provide a diagnosis in only a modest proportion of cases. The Full Genome Analysis method, FGA, combines long-range assembly and whole-genome sequencing to detect small variants, structural variants with breakpoint resolution, and phasing. We built a variant prioritization pipeline and tested FGA’s utility for diagnosis of rare diseases in a clinical setting. FGA identified structural variants and small variants with an overall diagnostic yield of 40% (20 of 50 cases) and 35% in exome-negative cases (8 of 23 cases), 4 of these were structural variants. FGA detected and mapped structural variants that are missed by short reads, including non-coding duplication, and phased variants across long distances of more than 180kb. With the prioritization algorithm, longer DNA technologies could replace multiple tests for monogenic disorders and expand the range of variants detected. Our study suggests that genomes produced from technologies like FGA can improve variant detection and provide higher resolution genome maps for future application.

Cenote-Taker 2 Democratizes Virus Discovery and Sequence Annotation

Viruses, despite their great abundance and significance in biological systems, remain largely mysterious. Indeed, the vast majority of the perhaps hundreds of millions of viral species on the planet remain undiscovered. Additionally, many viruses deposited in central databases like GenBank and RefSeq are littered with genes annotated as “hypothetical protein” or the equivalent. Cenote-Taker2, a virus discovery and annotation tool available on command line and with a graphical user interface with free high-performance computation access, utilizes highly sensitive models of hallmark virus genes to discover familiar or divergent viral sequences from user-input contigs. Additionally, Cenote-Taker2 uses a flexible set of modules to automatically annotate the sequence features of contigs, providing more gene information than comparable tools. The outputs include readable and interactive genome maps, virome summary tables, and files that can be directly submitted to GenBank. We expect Cenote-Taker2 to facilitate virus discovery, annotation, and expansion of the known virome.

Phigaro: high-throughput prophage sequence annotation

Summary Phigaro is a standalone command-line application that is able to detect prophage regions taking raw genome and metagenome assemblies as an input. It also produces dynamic annotated ‘prophage genome maps’ and marks possible transposon insertion spots inside prophages. It is applicable for mining prophage regions from large metagenomic datasets. Availability and implementation Source code for Phigaro is freely available for download at https://github.com/bobeobibo/phigaro along with test data. The code is written in Python. Supplementary information Supplementary data are available at Bioinformatics online.

Genome and Transcriptome Sequencing of casper and roy Zebrafish Mutants Provides Novel Genetic Clues for Iridophore Loss

casper has been a widely used transparent mutant of zebrafish. It possesses a combined loss of reflective iridophores and light-absorbing melanophores, which gives rise to its almost transparent trunk throughout larval and adult stages. Nevertheless, genomic causal mutations of this transparent phenotype are poorly defined. To identify the potential genetic basis of this fascinating morphological phenotype, we constructed genome maps by performing genome sequencing of 28 zebrafish individuals including wild-type AB strain, roy orbison (roy), and casper mutants. A total of 4.3 million high-quality and high-confidence homozygous single nucleotide polymorphisms (SNPs) were detected in the present study. We also identified a 6.0-Mb linkage disequilibrium block specifically in both roy and casper that was composed of 39 functional genes, of which the mpv17 gene was potentially involved in the regulation of iridophore formation and maintenance. This is the first report of high-confidence genomic mutations in the mpv17 gene of roy and casper that potentially leads to defective splicing as one major molecular clue for the iridophore loss. Additionally, comparative transcriptomic analyses of skin tissues from the AB, roy and casper groups revealed detailed transcriptional changes of several core genes that may be involved in melanophore and iridophore degeneration. In summary, our updated genome and transcriptome sequencing of the casper and roy mutants provides novel genetic clues for the iridophore loss. These new genomic variation maps will offer a solid genetic basis for expanding the zebrafish mutant database and in-depth investigation into pigmentation of animals.

New Tools for Hop Cytogenomics: Identification of Tandem Repeat Families from Long-Read Sequences of Humulus lupulus

ABSTRACTHop (Humulus lupulus L.) is known for its use as a bittering agent in beer and has a rich history of cultivation, beginning in Europe and now spanning the globe. There are five wild varieties worldwide, which may have been introgressed with cultivated varieties. As a dioecious species, its obligate outcrossing, non-Mendelian inheritance, and genomic structural variability have confounded directed breeding efforts. Consequently, understanding genome evolution in Humulus represents a considerable challenge, requiring additional resources, including integrated genome maps. In order to facilitate cytogenetic investigations into the transmission genetics of hop, we report here the identification and characterization of 17 new and distinct tandem repeat sequence families. A tandem repeat discovery pipeline was developed using k-mer filtering and dot plot analysis of PacBio long-read sequences from the hop cultivar Apollo. We produced oligonucleotide FISH probes from conserved regions of HuluTR120 and HulTR225 and demonstrated their utility to stain meiotic chromosomes from wild hop, var. neomexicanus. The HuluTR225 FISH probe hybridized to several loci per nucleus and exhibited irregular, non-Mendelian transmission in male meiocytes of wild hop. Collectively, these tandem repeat sequence families not only represent unique and valuable new cytogenetic reagents but also have the capacity to inform genome assembly efforts and support comparative genomic analyses.

RH mapping by sequencing: chromosome-scale assembly of the duck genome

Like many other species, the duck genome has been sequenced thanks to the technological breakthrough provided by the emergence of Next Generation Sequencing (NGS). The resulting de novo assemblies are however made of thousands of scattered scaffolds. To achieve chromosome-scale contiguity, long-range intermediate genome maps remain indispensable. Radiation Hybrid (RH) maps have been used to assist the generation of chromosome-scale genome assemblies by taking advantage of the high density SNP chips that provide a large number of markers that can be efficiently genotyped on the panel.In the absence of such a resource in duck, we sequenced 100 hybrid clones of a duck RH panel enabling direct genotyping of the assembly scaffolds on the panel. The rationale is to use scaffolds as markers and to genotype the scaffolds by sequencing the clones: the presence/absence of a scaffold in a particular sequenced hybrid is attested by the presence/absence of reads mapping specifically to this scaffold. The detection of scaffolds exhibiting a chromosomal breakage resulting from the irradiation process revealed itself to be a critical issue of this genotyping by sequencing process. This process resulted in the construction of RH vectors for 2,027 scaffolds, representing a total of about 1 Gb of sequences (95% of the current Duck genome assembly). The subsequent linkage analysis enabled the construction of RH maps and therefore to organize, i.e. order and orient, the scaffolds into pseudomolecules associated to the corresponding duck chromosomes. We describe here the whole mapping process, from sequence-based genotyping to the construction of comparative maps, as well as few examples of intra-chromosomal rearrangements that have been identified by the comparison with the chicken, turkey and zebra finch genomes and subsequently confirmed by FISH.We describe a method to order and orient sequence scaffolds into super-scaffolds spanning entire chromosomes. The method, which requires a pre-existing RH panel and sequence scaffolds from an NGS assembly, relies on a shallow sequencing of the RH clones. This approach was applied to the duck genome and produced chromosome-scale scaffolds for 29 out of the 41 duck chromosomes.

Genetics without genes? The centrality of genetic markers in livestock genetics and genomics

In this paper, rather than focusing on genes as an organising concept around which historical considerations of theory and practice in genetics are elucidated, we place genetic markers at the heart of our analysis. This reflects their central role in the subject of our account, livestock genetics concerning the domesticated pig, Sus scrofa. We define a genetic marker as a (usually material) element existing in different forms in the genome, that can be identified and mapped using a variety (and often combination) of quantitative, classical and molecular genetic techniques. The conjugation of pig genome researchers around the common object of the marker from the early-1990s allowed the distinctive theories and approaches of quantitative and molecular genetics concerning the size and distribution of gene effects to align (but never fully integrate) in projects to populate genome maps. Critical to this was the nature of markers as ontologically inert, internally heterogeneous and relational. Though genes as an organising and categorising principle remained important, the particular concatenation of limitations, opportunities, and intended research goals of the pig genetics community, meant that a progressively stronger focus on the identification and mapping of markers rather than genes per se became a hallmark of the community. We therefore detail a different way of doing genetics to more gene-centred accounts. By doing so, we reveal the presence of practices, concepts and communities that would otherwise be hidden.