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.…
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…
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,…
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.
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…
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…
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…
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…
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.
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),…
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…
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)…
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…
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.
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…