July 19, 2019  |  

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germ line and revealed somatic recombination. When the recombination partner was a diverged D. melanogaster genome or a genome from a different species such as D. yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.


July 19, 2019  |  

Major improvements to the Heliconius melpomene genome assembly used to confirm 10 chromosome fusion events in 6 million years of butterfly evolution.

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ~20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr. Copyright © 2016 Davey et al.


July 19, 2019  |  

Highly efficient CRISPR/Cas9-mediated cloning and functional characterization of gastric cancer-derived Epstein-Barr virus strains.

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death.Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 19, 2019  |  

Complete genome sequences of isolates of Enterococcus faecium sequence type 117, a globally disseminated multidrug-resistant clone.

The emergence of nosocomial infections by multidrug-resistant sequence type 117 (ST117) Enterococcus faecium has been reported in several European countries. ST117 has been detected in Spanish hospitals as one of the main causes of bloodstream infections. We analyzed genome variations of ST117 strains isolated in Madrid and describe the first ST117 closed genome sequences. Copyright © 2017 Tedim et al.


July 19, 2019  |  

Detecting AGG interruptions in male and female FMR1 premutation carriers by single-molecule sequencing.

The FMR1 gene contains an unstable CGG repeat in its 5′ untranslated region. Premutation alleles range between 55 and 200 repeat units and confer a risk for developing fragile X-associated tremor/ataxia syndrome or fragile X-associated primary ovarian insufficiency. Furthermore, the premutation allele often expands to a full mutation during female germline transmission giving rise to the fragile X syndrome. The risk for a premutation to expand depends mainly on the number of CGG units and the presence of AGG interruptions in the CGG repeat. Unfortunately, the detection of AGG interruptions is hampered by technical difficulties. Here, we demonstrate that single-molecule sequencing enables the determination of not only the repeat size, but also the complete repeat sequence including AGG interruptions in male and female alleles with repeats ranging from 45 to 100 CGG units. We envision this method will facilitate research and diagnostic analysis of the FMR1 repeat expansion. © 2016 WILEY PERIODICALS, INC.


July 19, 2019  |  

Analysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline.

The Lyme disease spirochete evades the host immune system by combinatorial variation of VlsE, a surface antigen. Antigenic variation occurs via segmental gene conversion from contiguous silent cassettes into the vlsE locus. Because of the high degree of similarity between switch variants and the size of vlsE, short-read NGS technologies have been unsuitable for sequencing vlsE populations. Here we use PacBio sequencing technology coupled with the first fully-automated software pipeline (VAST) to accurately process NGS data by minimizing error frequency, eliminating heteroduplex errors and accurately aligning switch variants. We extend earlier studies by showing use of almost all of the vlsE SNP repertoire. In different tissues of the same mouse, 99.6% of the variants were unique, suggesting that dissemination of Borrelia burgdorferi is predominantly unidirectional with little tissue-to-tissue hematogenous dissemination. We also observed a similar number of variants in SCID and wild-type mice, a heatmap of location and frequency of amino acid changes on the 3D structure and note differences observed in SCID versus wild type mice that hint at possible amino acid function. Our observed selection against diversification of residues at the dimer interface in wild-type mice strongly suggests that dimerization is required for in vivo functionality of vlsE.© 2017 John Wiley & Sons Ltd.


July 19, 2019  |  

Pacific Biosciences sequencing and IMGT/HighV-QUEST analysis of full-length single chain fragment variable from an in vivo selected phage-display combinatorial Library.

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires bothin vivoandin vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800?bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from anin vivoselection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of thein vivoselection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during thein vivopanning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767?bp, 53 of them covering the whole insert of 977?bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939?bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.


July 19, 2019  |  

Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema.

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is a-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a-e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research. Copyright © 2018 the Author(s). Published by PNAS.


July 19, 2019  |  

High-Throughput Single-Cell Sequencing of both TCR-ß Alleles.

Allelic exclusion is a vital mechanism for the generation of monospecificity to foreign Ags in B and T lymphocytes. In this study, we developed a high-throughput barcoded method to simultaneously analyze the VDJ recombination status of both mouse TCR-ß alleles in hundreds of single cells using next-generation sequencing. Copyright © 2018 by The American Association of Immunologists, Inc.


July 19, 2019  |  

Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: One species, four names.

We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species.


July 19, 2019  |  

Characterization of a human-specific tandem repeat associated with bipolar disorder and schizophrenia.

Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect more than 3% of individuals worldwide. Genome-wide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a human-specific tandem repeat in this region that is composed of 30 bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single-nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution and may modulate neuropsychiatric disease risk in modern human populations. Copyright © 2018. Published by Elsevier Inc.


July 7, 2019  |  

Common cell shape evolution of two nasopharyngeal pathogens.

Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.


July 7, 2019  |  

Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates in a university hospital in Nepal reveals the emergence of a novel epidemic clonal lineage.

The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious medical problem worldwide. To clarify the genetic and epidemiological properties of MDR A. baumannii strains isolated from a medical setting in Nepal, 246 Acinetobacter spp. isolates obtained from different patients were screened for MDR A. baumannii by antimicrobial disk susceptibility testing. Whole genomes of the MDR A. baumannii isolates were sequenced by MiSeq™ (Illumina), and the complete genome of one isolate (IOMTU433) was sequenced by PacBio RS II. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Of the 246 Acinetobacter spp. isolates, 122 (49.6%) were MDR A. baumannii, with the majority being resistant to aminoglycosides, carbapenems and fluoroquinolones but not to colistin and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as bla(NDM-1), bla(OXA-23) or bla(OXA-58). MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured genes encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA). Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.


July 7, 2019  |  

Strain Kaplan of pseudorabies virus genome sequenced by PacBio single-molecule real-time sequencing technology.

Pseudorabies virus (PRV) is a neurotropic herpesvirus that causes Aujeszky’s disease in pigs. PRV strains are widely used as transsynaptic tracers for mapping neural circuits. We present here the complete and fully annotated genome sequence of strain Kaplan of PRV, determined by Pacific Biosciences RSII long-read sequencing technology. Copyright © 2014 Tombácz et al.


July 7, 2019  |  

Enhancing the detection of barcoded reads in high throughput DNA sequencing data by controlling the false discovery rate.

DNA barcodes are short unique sequences used to label DNA or RNA-derived samples in multiplexed deep sequencing experiments. During the demultiplexing step, barcodes must be detected and their position identified. In some cases (e.g., with PacBio SMRT), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements.In our analysis, barcode sequences showed high rates of coincidental similarities with the Mus musculus reference DNA. This problem became more acute when the length of the barcode sequence decreased and the number of barcodes in the set increased. The method presented in this paper controls the tail area-based false discovery rate to distinguish between barcoded and unbarcoded reads. This method helps to establish the highest acceptable minimal distance between reads and barcode sequences. In a proof of concept experiment we correctly detected barcodes in 83% of the reads with a precision of 89%. Sensitivity improved to 99% at 99% precision when the adjacent primer sequence was incorporated in the analysis. The analysis was further improved using a paired end strategy. Following an analysis of the data for sequence variants induced in the Atp1a1 gene of C57BL/6 murine melanocytes by ultraviolet light and conferring resistance to ouabain, we found no evidence of cross-contamination of DNA material between samples.Our method offers a proper quantitative treatment of the problem of detecting barcoded reads in a noisy sequencing environment. It is based on the false discovery rate statistics that allows a proper trade-off between sensitivity and precision to be chosen.


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