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April 21, 2020  |  

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.


April 21, 2020  |  

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.


April 21, 2020  |  

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.


April 21, 2020  |  

Long-Read RNA Sequencing Identifies Alternative Splice Variants in Hepatocellular Carcinoma and Tumor-Specific Isoforms.

Alternative splicing (AS) allows generation of cell type-specific mRNA transcripts and contributes to hallmarks of cancer. Genome-wide analysis for AS in human hepatocellular carcinoma (HCC), however, is limited. We sought to obtain a comprehensive AS landscape in HCC and define tumor-associated variants. Single-molecule real-time long-read RNA sequencing was performed on patient-derived HCC cells, and presence of splice junctions was defined by SpliceMap-LSC-IDP algorithm. We obtained an all-inclusive map of annotated AS variants and further discovered 362 alternative spliced variants that are not previously reported in any database (neither RefSeq nor GENCODE). They were mostly derived from intron retention and early termination codon with an in-frame open reading frame in 81.5%. We corroborated many of these predicted unannotated and annotated variants to be tumor specific in an independent cohort of primary HCC tumors and matching nontumoral liver. Using the combined Sanger sequencing and TaqMan junction assays, unique and common expressions of spliced variants including enzyme regulators (ARHGEF2, SERPINH1), chromatin modifiers (DEK, CDK9, RBBP7), RNA-binding proteins (SRSF3, RBM27, MATR3, YBX1), and receptors (ADRM1, CD44v8-10, vitamin D receptor, ROR1) were determined in HCC tumors. We further focused functional investigations on ARHGEF2 variants (v1 and v3) that arise from the common amplified site chr.1q22 of HCC. Their biological significance underscores two major cancer hallmarks, namely cancer stemness and epithelial-to-mesenchymal transition-mediated cell invasion and migration, although v3 is consistently more potent than v1. Conclusion: Alternative isoforms and tumor-specific isoforms that arise from aberrant splicing are common during the liver tumorigenesis. Our results highlight insights gained from the analysis of AS in HCC. © 2019 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.


April 21, 2020  |  

The genomes of pecan and Chinese hickory provide insights into Carya evolution and nut nutrition.

Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and B vitamins. Further analyses of reduced-coverage genome sequences of 16 Carya and 2 Juglans species provide additional phylogenetic perspective on crop wild relatives.Cooperative characterization of these valuable resources provides a window to their evolutionary development and a valuable foundation for future crop improvement. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

The Modern View of B Chromosomes Under the Impact of High Scale Omics Analyses.

Supernumerary B chromosomes (Bs) are extra karyotype units in addition to A chromosomes, and are found in some fungi and thousands of animals and plant species. Bs are uniquely characterized due to their non-Mendelian inheritance, and represent one of the best examples of genomic conflict. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. A classical concept based on cytogenetics and genetics is that Bs are selfish and abundant with DNA repeats and transposons, and in most cases, they do not carry any function. However, recently, the modern quantum development of high scale multi-omics techniques has shifted B research towards a new-born field that we call “B-omics”. We review the recent literature and add novel perspectives to the B research, discussing the role of new technologies to understand the mechanistic perspectives of the molecular evolution and function of Bs. The modern view states that B chromosomes are enriched with genes for many significant biological functions, including but not limited to the interesting set of genes related to cell cycle and chromosome structure. Furthermore, the presence of B chromosomes could favor genomic rearrangements and influence the nuclear environment affecting the function of other chromatin regions. We hypothesize that B chromosomes might play a key function in driving their transmission and maintenance inside the cell, as well as offer an extra genomic compartment for evolution.


April 21, 2020  |  

A Genome-Wide Epstein-Barr Virus Polyadenylation Map and Its Antisense RNA to EBNA.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen associated with Burkitt’s lymphoma and nasopharyngeal carcinoma. Although the EBV genome harbors more than a hundred genes, a full transcription map with EBV polyadenylation profiles remains unknown. To elucidate the 3′ ends of all EBV transcripts genome-wide, we performed the first comprehensive analysis of viral polyadenylation sites (pA sites) using our previously reported polyadenylation sequencing (PA-seq) technology. We identified that EBV utilizes a total of 62?pA sites in JSC-1, 60 in Raji, and 53 in Akata cells for the expression of EBV genes from both plus and minus DNA strands; 42 of these pA sites are commonly used in all three cell lines. The majority of identified pA sites were mapped to the intergenic regions downstream of previously annotated EBV open reading frames (ORFs) and viral promoters. pA sites lacking an association with any known EBV genes were also identified, mostly for the minus DNA strand within the EBNA locus, a major locus responsible for maintenance of viral latency and cell transformation. The expression of these novel antisense transcripts to EBNA were verified by 3′ rapid amplification of cDNA ends (RACE) and Northern blot analyses in several EBV-positive (EBV+) cell lines. In contrast to EBNA RNA expressed during latency, expression of EBNA-antisense transcripts, which is restricted in latent cells, can be significantly induced by viral lytic infection, suggesting potential regulation of viral gene expression by EBNA-antisense transcription during lytic EBV infection. Our data provide the first evidence that EBV has an unrecognized mechanism that regulates EBV reactivation from latency.IMPORTANCE Epstein-Barr virus represents an important human pathogen with an etiological role in the development of several cancers. By elucidation of a genome-wide polyadenylation landscape of EBV in JSC-1, Raji, and Akata cells, we have redefined the EBV transcriptome and mapped individual polymerase II (Pol II) transcripts of viral genes to each one of the mapped pA sites at single-nucleotide resolution as well as the depth of expression. By unveiling a new class of viral lytic RNA transcripts antisense to latent EBNAs, we provide a novel mechanism of how EBV might control the expression of viral latent genes and lytic infection. Thus, this report takes another step closer to understanding EBV gene structure and expression and paves a new path for antiviral approaches.This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.


April 21, 2020  |  

Gammaherpesvirus Readthrough Transcription Generates a Long Non-Coding RNA That Is Regulated by Antisense miRNAs and Correlates with Enhanced Lytic Replication In Vivo.

Gammaherpesviruses, including the human pathogens Epstein?Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, ?HV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the M3/M2 locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of M3-04, a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two M3-04-antisense pre-miRNA stem loops resulted in highly increased expression of M3-04 and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA M3-04, antisense miRNAs, and the latency gene M2.


April 21, 2020  |  

Diffusely Adherent Escherichia coli Strains Isolated from Healthy Carriers Suppress Cytokine Secretions of Epithelial Cells Stimulated by Inflammatory Substances.

Diarrheagenicity of diffusely adherent Escherichia coli (DAEC) remains controversial. Previously, we found that motile DAEC strains isolated from diarrheal patients induced high levels of interleukin 8 (IL-8) secretion via Toll-like receptor 5 (TLR5). However, DAEC strains from healthy carriers hardly induced IL-8 secretion, irrespective of their possessing flagella. In this study, we demonstrated that SK1144, a DAEC strain from a healthy carrier, suppressed IL-8 and IL-6 secretion from human epithelial cell lines. Suppression of IL-8 in human embryonic kidney (HEK293) cells that were transformed to express TLR5 was observed not only upon inflammatory stimulation by flagellin but also in response to tumor necrosis factor alpha (TNF-a) and phorbol myristate acetate (PMA), despite the fact that the TNF-a- and PMA-induced inflammatory pathways reportedly are not TLR5 mediated. SK1144 neither decreased IL-8 transcript accumulation nor increased intracellular retention of IL-8. No suppression was observed when the bacteria were cultured in Transwell cups above the epithelial cells; however, a nonadherent bacterial mutant (lacking the afimbrial adhesin gene) still inhibited IL-8 secretion. Direct contact between the bacteria and epithelial cells was necessary, but diffuse adhesion was dispensable for the inhibitory effects. Infection in the presence of chloramphenicol did not suppress cytokine release by the epithelial cells, suggesting that suppression depended on effectors synthesized de novo Inflammatory suppression was attenuated with infection by a bacterial mutant deleted for hcp (encoding a component of a type VI secretion system). In conclusion, DAEC strains from healthy carriers impede epithelial cell cytokine secretion, possibly by interfering with translation via the type VI secretion system.Copyright © 2018 American Society for Microbiology.


April 21, 2020  |  

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes

As they migrated out of Africa and into Europe and Asia, anatomically modern humans interbred with archaic hominins, such as Neanderthals and Denisovans. The result of this genetic introgression on the recipient populations has been of considerable interest, especially in cases of selection for specific archaic genetic variants. Hsieh et al. characterized adaptive structural variants and copy number variants that are likely targets of positive selection in Melanesians. Focusing on population-specific regions of the genome that carry duplicated genes and show an excess of amino acid replacements provides evidence for one of the mechanisms by which genetic novelty can arise and result in differentiation between human genomes.Science, this issue p. eaax2083INTRODUCTIONCharacterizing genetic variants underlying local adaptations in human populations is one of the central goals of evolutionary research. Most studies have focused on adaptive single-nucleotide variants that either arose as new beneficial mutations or were introduced after interbreeding with our now-extinct relatives, including Neanderthals and Denisovans. The adaptive role of copy number variants (CNVs), another well-known form of genomic variation generated through deletions or duplications that affect more base pairs in the genome, is less well understood, despite evidence that such mutations are subject to stronger selective pressures.RATIONALEThis study focuses on the discovery of introgressed and adaptive CNVs that have become enriched in specific human populations. We combine whole-genome CNV calling and population genetic inference methods to discover CNVs and then assess signals of selection after controlling for demographic history. We examine 266 publicly available modern human genomes from the Simons Genome Diversity Project and genomes of three ancient homininstextemdasha Denisovan, a Neanderthal from the Altai Mountains in Siberia, and a Neanderthal from Croatia. We apply long-read sequencing methods to sequence-resolve complex CNVs of interest specifically in the Melanesianstextemdashan Oceanian population distributed from Papua New Guinea to as far east as the islands of Fiji and known to harbor some of the greatest amounts of Neanderthal and Denisovan ancestry.RESULTSConsistent with the hypothesis of archaic introgression outside Africa, we find a significant excess of CNV sharing between modern non-African populations and archaic hominins (P = 0.039). Among Melanesians, we observe an enrichment of CNVs with potential signals of positive selection (n = 37 CNVs), of which 19 CNVs likely introgressed from archaic hominins. We show that Melanesian-stratified CNVs are significantly associated with signals of positive selection (P = 0.0323). Many map near or within genes associated with metabolism (e.g., ACOT1 and ACOT2), development and cell cycle or signaling (e.g., TNFRSF10D and CDK11A and CDK11B), or immune response (e.g., IFNLR1). We characterize two of the largest and most complex CNVs on chromosomes 16p11.2 and 8p21.3 that introgressed from Denisovans and Neanderthals, respectively, and are absent from most other human populations. At chromosome 16p11.2, we sequence-resolve a large duplication of >383 thousand base pairs (kbp) that originated from Denisovans and introgressed into the ancestral Melanesian population 60,000 to 170,000 years ago. This large duplication occurs at high frequency (>79%) in diverse Melanesian groups, shows signatures of positive selection, and maps adjacent to Homo sapienstextendashspecific duplications that predispose to rearrangements associated with autism. On chromosome 8p21.3, we identify a Melanesian haplotype that carries two CNVs, a ~6-kbp deletion, and a ~38-kbp duplication, with a Neanderthal origin and that introgressed into non-Africans 40,000 to 120,000 years ago. This CNV haplotype occurs at high frequency (44%) and shows signals consistent with a partial selective sweep in Melanesians. Using long-read sequencing genomic and transcriptomic data, we reconstruct the structure and complex evolutionary history for these two CNVs and discover previously undescribed duplicated genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that show an excess of amino acid replacements consistent with the action of positive selection.CONCLUSIONOur results suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation that is absent from current reference genomes.Large adaptive-introgressed CNVs at chromosomes 8p21.3 and 16p11.2 in Melanesians.The magnifying glasses highlight structural differences between the archaic (top) and reference (bottom) genomes. Neanderthal (red) and Denisovan (blue) haplotypes encompassing large CNVs occur at high frequencies in Melanesians (44 and 79%, respectively) but are absent (black) in all non-Melanesians. These CNVs create positively selected genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that are absent from the reference genome.Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.


April 21, 2020  |  

A personalized platform identifies trametinib plus zoledronate for a patient with KRAS-mutant metastatic colorectal cancer.

Colorectal cancer remains a leading source of cancer mortality worldwide. Initial response is often followed by emergent resistance that is poorly responsive to targeted therapies, reflecting currently undruggable cancer drivers such as KRAS and overall genomic complexity. Here, we report a novel approach to developing a personalized therapy for a patient with treatment-resistant metastatic KRAS-mutant colorectal cancer. An extensive genomic analysis of the tumor’s genomic landscape identified nine key drivers. A transgenic model that altered orthologs of these nine genes in the Drosophila hindgut was developed; a robotics-based screen using this platform identified trametinib plus zoledronate as a candidate treatment combination. Treating the patient led to a significant response: Target and nontarget lesions displayed a strong partial response and remained stable for 11 months. By addressing a disease’s genomic complexity, this personalized approach may provide an alternative treatment option for recalcitrant disease such as KRAS-mutant colorectal cancer.


April 21, 2020  |  

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.


April 21, 2020  |  

FadR1, a pathway-specific activator of fidaxomicin biosynthesis in Actinoplanes deccanensis Yp-1.

Fidaxomicin, an 18-membered macrolide antibiotic, is highly active against Clostridium difficile, the most common cause of diarrhea in hospitalized patients. Though the biosynthetic mechanism of fidaxomicin has been well studied, little is known about its regulatory mechanism. Here, we reported that FadR1, a LAL family transcriptional regulator in the fidaxomicin cluster of Actinoplanes deccanensis Yp-1, acts as an activator for fidaxomicin biosynthesis. The disruption of fadR1 abolished the ability to synthesize fidaxomicin, and production could be restored by reintegrating a single copy of fadR1. Overexpression of fadR1 resulted in an approximately 400 % improvement in fidaxomicin production. Electrophoretic mobility shift assays indicated that fidaxomicin biosynthesis is under the control of FadR1 through its binding to the promoter regions of fadM, fadA1-fadP2, fadS2-fadC, and fadE-fadF, respectively. And the conserved binding sites of FadR1 within the four promoter regions were determined by footprinting experiment. All results indicated that fadR1 encodes a pathway-specific positive regulator of fidaxomicin biosynthesis and upregulates the transcription levels of most of genes by binding to the four above intergenic regions. In summary, we not only clearly elucidate the regulatory mechanism of FadR1 but also provide strategies for the construction of industrial high-yield strain of fidaxomicin.


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