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September 22, 2019

Improved reference genome for the domestic horse increases assembly contiguity and composition.

Recent advances in genomic sequencing technology and computational assembly methods have allowed scientists to improve reference genome assemblies in terms of contiguity and composition. EquCab2, a reference genome for the domestic horse, was released in 2007. Although of equal or better quality compared to other first-generation Sanger assemblies, it had many of the shortcomings common to them. In 2014, the equine genomics research community began a project to improve the reference sequence for the horse, building upon the solid foundation of EquCab2 and incorporating new short-read data, long-read data, and proximity ligation data. Here, we present EquCab3. The count of non-N bases in the incorporated chromosomes is improved from 2.33?Gb in EquCab2 to 2.41?Gb in EquCab3. Contiguity has also been improved nearly 40-fold with a contig N50 of 4.5?Mb and scaffold contiguity enhanced to where all but one of the 32 chromosomes is comprised of a single scaffold.

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September 22, 2019

Loss of Rap1 supports recombination-based telomere maintenance independent of RNA-DNA hybrids in fission yeast

To investigate the molecular changes needed for cells to maintain their telomeres by recombination, we monitored telomere appearance during serial culture of fission yeast cells lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). Degradation of telomere-associated TERRA in this context drives a severe growth crisis, ultimately leading to a distinct type of linear survivor with altered cytological telomere characteristics and the eviction of the shelterin component Rap1 (but not the TRF1/TRF2 orthologue, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective, preventing the growth crisis that is otherwise triggered by degradation of telomere-engaged TERRA in survivors with linear chromosomes. Thus, modulating the stoichiometry of shelterin components appears to support recombination-dependent survivors to persist in the absence of telomere-engaged TERRA.

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September 22, 2019

Evolutionary conservation of Y Chromosome ampliconic gene families despite extensive structural variation.

Despite claims that the mammalian Y Chromosome is on a path to extinction, comparative sequence analysis of primate Y Chromosomes has shown the decay of the ancestral single-copy genes has all but ceased in this eutherian lineage. The suite of single-copy Y-linked genes is highly conserved among the majority of eutherian Y Chromosomes due to strong purifying selection to retain dosage-sensitive genes. In contrast, the ampliconic regions of the Y Chromosome, which contain testis-specific genes that encode the majority of the transcripts on eutherian Y Chromosomes, are rapidly evolving and are thought to undergo species-specific turnover. However, ampliconic genes are known from only a handful of species, limiting insights into their long-term evolutionary dynamics. We used a clone-based sequencing approach employing both long- and short-read sequencing technologies to assemble ~2.4 Mb of representative ampliconic sequence dispersed across the domestic cat Y Chromosome, and identified the major ampliconic gene families and repeat units. We analyzed fluorescence in situ hybridization, qPCR, and whole-genome sequence data from 20 cat species and revealed that ampliconic gene families are conserved across the cat family Felidae but show high transcript diversity, copy number variation, and structural rearrangement. Our analysis of ampliconic gene evolution unveils a complex pattern of long-term gene content stability despite extensive structural variation on a nonrecombining background.© 2018 Brashear et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Genetics and genomics of an unusual selfish sex ratio distortion in an insect.

Diverse selfish genetic elements have evolved the ability to manipulate reproduction to increase their transmission, and this can result in highly distorted sex ratios [1]. Indeed, one of the major explanations for why sex determination systems are so dynamic is because they are shaped by ongoing coevolutionary arms races between sex-ratio-distorting elements and the rest of the genome [2]. Here, we use genetic crosses and genome analysis to describe an unusual sex ratio distortion with striking consequences on genome organization in a booklouse species, Liposcelis sp. (Insecta: Psocodea), in which two types of females coexist. Distorter females never produce sons but must mate with males (the sons of nondistorting females) to reproduce [3]. Although they are diploid and express the genes inherited from their fathers in somatic tissues, distorter females only ever transmit genes inherited from their mothers. As a result, distorter females have unusual chimeric genomes, with distorter-restricted chromosomes diverging from their nondistorting counterparts and exhibiting features of a giant non-recombining sex chromosome. The distorter-restricted genome has also acquired a gene from the bacterium Wolbachia, a well-known insect reproductive manipulator; we found that this gene has independently colonized the genomes of two other insect species with unusual reproductive systems, suggesting possible roles in sex ratio distortion in this remarkable genetic system. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Sex chromosome evolution via two genes

The origin of sex chromosomes has been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (Asparagus officinalis L.) is an ideal species to test this hypothesis, as the X and Y chromosomes are cytologically homomorphic and recently evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with single-molecule fluorescence in situ hybridization (smFISH) directly implicate Y-specific genes that respectively suppress female organ development and are necessary for male gametophyte development. Comparison of contiguous X and Y chromosome shows that loss of recombination between the genes suppressing female function (SUPPRESSOR OF FEMALE FUNCTION, SOFF) and promoting male function (TAPETAL DEVELOPMENT AND FUNCTION 1, aspTDF1) is due to hemizygosity. We also experimentally demonstrate the function of aspTDF1. These finding provide direct evidence that sex chromosomes can evolve from autosomes via two sex determination genes: a dominant suppressor of femaleness and a promoter of maleness.

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September 21, 2019

Identification of a novel RASD1 somatic mutation in a USP8-mutated corticotroph adenoma.

Cushing’s disease (CD) is caused by pituitary corticotroph adenomas that secrete excess adrenocorticotropic hormone (ACTH). In these tumors, somatic mutations in the gene USP8 have been identified as recurrent and pathogenic and are the sole known molecular driver for CD. Although other somatic mutations were reported in these studies, their contribution to the pathogenesis of CD remains unexplored. No molecular drivers have been established for a large proportion of CD cases and tumor heterogeneity has not yet been investigated using genomics methods. Also, even in USP8-mutant tumors, a possibility may exist of additional contributing mutations, following a paradigm from other neoplasm types where multiple somatic alterations contribute to neoplastic transformation. The current study utilizes whole-exome discovery sequencing on the Illumina platform, followed by targeted amplicon-validation sequencing on the Pacific Biosciences platform, to interrogate the somatic mutation landscape in a corticotroph adenoma resected from a CD patient. In this USP8-mutated tumor, we identified an interesting somatic mutation in the gene RASD1, which is a component of the corticotropin-releasing hormone receptor signaling system. This finding may provide insight into a novel mechanism involving loss of feedback control to the corticotropin-releasing hormone receptor and subsequent deregulation of ACTH production in corticotroph tumors.

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September 21, 2019

Detecting AGG interruptions in females with a FMR1 premutation by long-read Single-Molecule Sequencing: A 1 year clinical experience.

The fragile X syndrome arises from the FMR1 CGG expansion of a premutation (55-200 repeats) to a full mutation allele (>200 repeats) and is the most frequent cause of inherited X-linked intellectual disability. The risk for a premutation to expand to a full mutation allele depends on the repeat length and AGG triplets interrupting this repeat. In genetic counseling it is important to have information on both these parameters to provide an accurate risk estimate to women carrying a premutation allele and weighing up having children. For example, in case of a small risk a woman might opt for a natural pregnancy followed up by prenatal diagnosis while she might choose for preimplantation genetic diagnosis (PGD) if the risk is high. Unfortunately, the detection of AGG interruptions was previously hampered by technical difficulties complicating their use in diagnostics. Therefore we recently developed, validated and implemented a new methodology which uses long-read single-molecule sequencing to identify AGG interruptions in females with a FMR1 premutation. Here we report on the assets of AGG interruption detection by sequencing and the impact of implementing the assay on genetic counseling.

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September 21, 2019

Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

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