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September 21, 2019

Divergent selection causes whole genome differentiation without physical linkage among the targets in Spodoptera frugiperda (Noctuidae)

The process of speciation involves whole genome differentiation by overcoming gene flow between diverging populations. We have ample knowledge which evolutionary forces may cause genomic differentiation, and several speciation models have been proposed to explain the transition from genetic to genomic differentiation. However, it is still unclear what are critical conditions enabling genomic differentiation in nature. The Fall armyworm, Spodoptera frugiperda, is observed as two sympatric strains that have different host-plant ranges, suggesting the possibility of ecological divergent selection. In our previous study, we observed that these two strains show genetic differentiation across the whole genome with an unprecedentedly low extent, suggesting the possibility that whole genome sequences started to be differentiated between the strains. In this study, we analyzed whole genome sequences from these two strains from Mississippi to identify critical evolutionary factors for genomic differentiation. The genomic Fst is low (0.017) while 91.3% of 10kb windows have Fst greater than 0, suggesting genome-wide differentiation with a low extent. We identified nearly 400 outliers of genetic differentiation between strains, and found that physical linkage among these outliers is not a primary cause of genomic differentiation. Fst is not significantly correlated with gene density, a proxy for the strength of selection, suggesting that a genomic reduction in migration rate dominates the extent of local genetic differentiation. Our analyses reveal that divergent selection alone is sufficient to generate genomic differentiation, and any following diversifying factors may increase the level of genetic differentiation between diverging strains in the process of speciation.

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September 21, 2019

Phased diploid genome assembly with single-molecule real-time sequencing.

While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.

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September 21, 2019

Discovery and genotyping of structural variation from long-read haploid genome sequence data.

In an effort to more fully understand the full spectrum of human genetic variation, we generated deep single-molecule, real-time (SMRT) sequencing data from two haploid human genomes. By using an assembly-based approach (SMRT-SV), we systematically assessed each genome independently for structural variants (SVs) and indels resolving the sequence structure of 461,553 genetic variants from 2 bp to 28 kbp in length. We find that >89% of these variants have been missed as part of analysis of the 1000 Genomes Project even after adjusting for more common variants (MAF > 1%). We estimate that this theoretical human diploid differs by as much as ~16 Mbp with respect to the human reference, with long-read sequencing data providing a fivefold increase in sensitivity for genetic variants ranging in size from 7 bp to 1 kbp compared with short-read sequence data. Although a large fraction of genetic variants were not detected by short-read approaches, once the alternate allele is sequence-resolved, we show that 61% of SVs can be genotyped in short-read sequence data sets with high accuracy. Uncoupling discovery from genotyping thus allows for the majority of this missed common variation to be genotyped in the human population. Interestingly, when we repeat SV detection on a pseudodiploid genome constructed in silico by merging the two haploids, we find that ~59% of the heterozygous SVs are no longer detected by SMRT-SV. These results indicate that haploid resolution of long-read sequencing data will significantly increase sensitivity of SV detection.© 2017 Huddleston et al.; Published by Cold Spring Harbor Laboratory Press.

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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

Long-read genome sequencing identifies causal structural variation in a Mendelian disease.

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions?>?50?bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184?bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.

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September 21, 2019

Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar Chardonnay.

Chardonnay is the basis of some of the world’s most iconic wines and its success is underpinned by a historic program of clonal selection. There are numerous clones of Chardonnay available that exhibit differences in key viticultural and oenological traits that have arisen from the accumulation of somatic mutations during centuries of asexual propagation. However, the genetic variation that underlies these differences remains largely unknown. To address this knowledge gap, a high-quality, diploid-phased Chardonnay genome assembly was produced from single-molecule real time sequencing, and combined with re-sequencing data from 15 different Chardonnay clones. There were 1620 markers identified that distinguish the 15 clones. These markers were reliably used for clonal identification of independently sourced genomic material, as well as in identifying a potential genetic basis for some clonal phenotypic differences. The predicted parentage of the Chardonnay haplomes was elucidated by mapping sequence data from the predicted parents of Chardonnay (Gouais blanc and Pinot noir) against the Chardonnay reference genome. This enabled the detection of instances of heterosis, with differentially-expanded gene families being inherited from the parents of Chardonnay. Most surprisingly however, the patterns of nucleotide variation present in the Chardonnay genome indicate that Pinot noir and Gouais blanc share an extremely high degree of kinship that has resulted in the Chardonnay genome displaying characteristics that are indicative of inbreeding.

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