Mutations causing medullary cystic kidney disease type 1 (MCKD1) lie in a large VNTR in MUC1 missed by massively parallel sequencing

While genetic lesions responsible for some Mendelian disorders can be rapidly discovered through massively parallel sequencing (MPS) of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple Mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing, and de novo assembly, we found that each of six MCKD1 families harbors an equivalent, but apparently independently arising, mutation in sequence dramatically underrepresented in MPS data: the insertion of a single C in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5-5 kb), GC-rich (>80%), coding VNTR in the mucin 1 gene. The results provide a cautionary tale about the challenges in identifying genes responsible for Mendelian, let alone more complex, disorders through MPS

Mutations causing medullary cystic kidney disease type 1 (MCKD1) lie in a large VNTR in MUC1 missed by massively parallel sequencing

While genetic lesions responsible for some Mendelian disorders can be rapidly discovered through massively parallel sequencing (MPS) of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple Mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing, and de novo assembly, we found that each of six MCKD1 families harbors an equivalent, but apparently independently arising, mutation in sequence dramatically underrepresented in MPS data: the insertion of a single C in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5-5 kb), GC-rich (>80%), coding VNTR in the mucin 1 gene. The results provide a cautionary tale about the challenges in identifying genes responsible for Mendelian, let alone more complex, disorders through MPS

EST Analysis of Ostreococcus lucimarinus, the Most Compact Eukaryotic Genome, Shows an Excess of Introns in Highly Expressed Genes

Background: The genome of the pico-eukaryotic (bacterial-sized) prasinophyte green alga Ostreococcus lucimarinus has one of the highest gene densities known in eukaryotes, yet it contains many introns. Phylogenetic studies suggest this unusually compact genome (13.2 Mb) is an evolutionarily derived state among prasinophytes. The presence of introns in the highly reduced O. lucimarinus genome appears to be in opposition to simple explanations of genome evolution based on unidirectional tendencies, either neutral or selective. Therefore, patterns of intron retention in this species can potentially provide insights into the forces governing intron evolution. Methodology/Principal Findings: Here we studied intron features and levels of expression in O. lucimarinus using expressed sequence tags (ESTs) to annotate the current genome assembly. ESTs were assembled into unigene clusters that were mapped back to the O. lucimarinus Build 2.0 assembly using BLAST and the level of gene expression was inferred from the number of ESTs in each cluster. We find a positive correlation between expression levels and both intron number (R = +0.0893, p =,0.0005) and intron density (number of introns/kb of CDS; R = +0.0753, p =,0.005). Conclusions/Significance: In a species with a genome that has been recently subjected to a great reduction of non-coding DNA, these results imply the existence of selective/functional roles for introns that are principally detectable in highly expressed genes. In these cases, introns are likely maintained by balancing the selective forces favoring their maintenanc

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity, using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two prostate cancer patients including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were observed in matched tissue. Moreover, we identified 10 early-trunk and 56 metastatic-trunk mutations in the non-CTC tumor samples and found 90% and 73% of these, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic

Mutations causing medullary cystic kidney disease type 1 lie in a large VNTR in MUC1 missed by massively parallel sequencing

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5–5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing.National Institutes of Health (U.S.) (Intramural Research Program)National Human Genome Research Institute (U.S.)Charles University (program UNCE 204011)Charles University (program PRVOUK-P24/LF1/3)Czech Republic. Ministry of Education, Youth, and Sports (grant NT13116-4/2012)Czech Republic. Ministry of Health (grant NT13116-4/2012)Czech Republic. Ministry of Health (grant LH12015)National Institutes of Health (U.S.) (Harvard Digestive Diseases Center, grant DK34854

Evolutionary Dynamics of the Ty3/Gypsy LTR Retrotransposons in the Genome of Anopheles gambiae

Ty3/gypsy elements represent one of the most abundant and diverse LTR-retrotransposon (LTRr) groups in the Anopheles gambiae genome, but their evolutionary dynamics have not been explored in detail. Here, we conduct an in silico analysis of the distribution and abundance of the full complement of 1045 copies in the updated AgamP3 assembly. Chromosomal distribution of Ty3/gypsy elements is inversely related to arm length, with densities being greatest on the X, and greater on the short versus long arms of both autosomes. Taking into account the different heterochromatic and euchromatic compartments of the genome, our data suggest that the relative abundance of Ty3/gypsy LTRrs along each chromosome arm is determined mainly by the different proportions of heterochromatin, particularly pericentric heterochromatin, relative to total arm length. Additionally, the breakpoint regions of chromosomal inversion 2La appears to be a haven for LTRrs. These elements are underrepresented more than 7-fold in euchromatin, where 33% of the Ty3/gypsy copies are associated with genes. The euchromatin on chromosome 3R shows a faster turnover rate of Ty3/gypsy elements, characterized by a deficit of proviral sequences and the lowest average sequence divergence of any autosomal region analyzed in this study. This probably reflects a principal role of purifying selection against insertion for the preservation of longer conserved syntenyc blocks with adaptive importance located in 3R. Although some Ty3/gypsy LTRrs show evidence of recent activity, an important fraction are inactive remnants of relatively ancient insertions apparently subject to genetic drift. Consistent with these computational predictions, an analysis of the occupancy rate of putatively older insertions in natural populations suggested that the degenerate copies have been fixed across the species range in this mosquito, and also are shared with the sibling species Anopheles arabiensis

Sequencing of Pooled DNA Samples (Pool-Seq) Uncovers Complex Dynamics of Transposable Element Insertions in Drosophila melanogaster

Transposable elements (TEs) are mobile genetic elements that parasitize genomes by semi-autonomously increasing their own copy number within the host genome. While TEs are important for genome evolution, appropriate methods for performing unbiased genome-wide surveys of TE variation in natural populations have been lacking. Here, we describe a novel and cost-effective approach for estimating population frequencies of TE insertions using paired-end Illumina reads from a pooled population sample. Importantly, the method treats insertions present in and absent from the reference genome identically, allowing unbiased TE population frequency estimates. We apply this method to data from a natural Drosophila melanogaster population from Portugal. Consistent with previous reports, we show that low recombining genomic regions harbor more TE insertions and maintain insertions at higher frequencies than do high recombining regions. We conservatively estimate that there are almost twice as many “novel” TE insertion sites as sites known from the reference sequence in our population sample (6,824 novel versus 3,639 reference sites, with on average a 31-fold coverage per insertion site). Different families of transposable elements show large differences in their insertion densities and population frequencies. Our analyses suggest that the history of TE activity significantly contributes to this pattern, with recently active families segregating at lower frequencies than those active in the more distant past. Finally, using our high-resolution TE abundance measurements, we identified 13 candidate positively selected TE insertions based on their high population frequencies and on low Tajima's D values in their neighborhoods

Production of Virus-Derived Ping-Pong-Dependent piRNA-like Small RNAs in the Mosquito Soma

The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication

The Epigenetic Trans-Silencing Effect in Drosophila Involves Maternally-Transmitted Small RNAs Whose Production Depends on the piRNA Pathway and HP1

BACKGROUND: The study of P transposable element repression in Drosophila melanogaster led to the discovery of the Trans-Silencing Effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, "TAS") has the capacity to repress in trans, in the female germline, a homologous P-lacZ transgene located in euchromatin. Phenotypic and genetic analysis have shown that TSE exhibits variegation in ovaries, displays a maternal effect as well as epigenetic transmission through meiosis and involves heterochromatin (including HP1) and RNA silencing. PRINCIPAL FINDINGS: Here, we show that mutations in squash and zucchini, which are involved in the piwi-interacting RNA (piRNA) silencing pathway, strongly affect TSE. In addition, we carried out a molecular analysis of TSE and show that silencing is correlated to the accumulation of lacZ small RNAs in ovaries. Finally, we show that the production of these small RNAs is sensitive to mutations affecting squash and zucchini, as well as to the dose of HP1. CONCLUSIONS AND SIGNIFICANCE: Thus, our results indicate that the TSE represents a bona fide piRNA-based repression. In addition, the sensitivity of TSE to HP1 dose suggests that in Drosophila, as previously shown in Schizosaccharomyces pombe, a RNA silencing pathway can depend on heterochromatin components

Holding it together: rapid evolution and positive selection in the synaptonemal complex of Drosophila

Background The synaptonemal complex (SC) is a highly conserved meiotic structure that functions to pair homologs and facilitate meiotic recombination in most eukaryotes. Five Drosophila SC proteins have been identified and localized within the complex: C(3)G, C(2)M, CONA, ORD, and the newly identified Corolla. The SC is required for meiotic recombination in Drosophila and absence of these proteins leads to reduced crossing over and chromosomal nondisjunction. Despite the conserved nature of the SC and the key role that these five proteins have in meiosis in D. melanogaster, they display little apparent sequence conservation outside the genus. To identify factors that explain this lack of apparent conservation, we performed a molecular evolutionary analysis of these genes across the Drosophila genus. Results For the five SC components, gene sequence similarity declines rapidly with increasing phylogenetic distance and only ORD and C(2)M are identifiable outside of the Drosophila genus. SC gene sequences have a higher dN/dS (ω) rate ratio than the genome wide average and this can in part be explained by the action of positive selection in almost every SC component. Across the genus, there is significant variation in ω for each protein. It further appears that ω estimates for the five SC components are in accordance with their physical position within the SC. Components interacting with chromatin evolve slowest and components comprising the central elements evolve the most rapidly. Finally, using population genetic approaches, we demonstrate that positive selection on SC components is ongoing. Conclusions SC components within Drosophila show little apparent sequence homology to those identified in other model organisms due to their rapid evolution. We propose that the Drosophila SC is evolving rapidly due to two combined effects. First, we propose that a high rate of evolution can be partly explained by low purifying selection on protein components whose function is to simply hold chromosomes together. We also propose that positive selection in the SC is driven by its sex-specificity combined with its role in facilitating both recombination and centromere clustering in the face of recurrent bouts of drive in female meiosis