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July 7, 2019

BreakSeek: a breakpoint-based algorithm for full spectral range INDEL detection.

Although recent developed algorithms have integrated multiple signals to improve sensitivity for insertion and deletion (INDEL) detection, they are far from being perfect and still have great limitations in detecting a full size range of INDELs. Here we present BreakSeek, a novel breakpoint-based algorithm, which can unbiasedly and efficiently detect both homozygous and heterozygous INDELs, ranging from several base pairs to over thousands of base pairs, with accurate breakpoint and heterozygosity rate estimations. Comprehensive evaluations on both simulated and real datasets revealed that BreakSeek outperformed other existing methods on both sensitivity and specificity in detecting both small and large INDELs, and uncovered a significant amount of novel INDELs that were missed before. In addition, by incorporating sophisticated statistic models, we for the first time investigated and demonstrated the importance of handling false and conflicting signals for multi-signal integrated methods.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

Comparative Analysis of the Shared Sex-Determination Region (SDR) among Salmonid Fishes.

Salmonids present an excellent model for studying evolution of young sex-chromosomes. Within the genus, Oncorhynchus, at least six independent sex-chromosome pairs have evolved, many unique to individual species. This variation results from the movement of the sex-determining gene, sdY, throughout the salmonid genome. While sdY is known to define sexual differentiation in salmonids, the mechanism of its movement throughout the genome has remained elusive due to high frequencies of repetitive elements, rDNA sequences, and transposons surrounding the sex-determining regions (SDR). Despite these difficulties, bacterial artificial chromosome (BAC) library clones from both rainbow trout and Atlantic salmon containing the sdY region have been reported. Here, we report the sequences for these BACs as well as the extended sequence for the known SDR in Chinook gained through genome walking methods. Comparative analysis allowed us to study the overlapping SDRs from three unique salmonid Y chromosomes to define the specific content, size, and variation present between the species. We found approximately 4.1 kb of orthologous sequence common to all three species, which contains the genetic content necessary for masculinization. The regions contain transposable elements that may be responsible for the translocations of the SDR throughout salmonid genomes and we examine potential mechanistic roles of each one. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Library construction for high-throughput mobile element identification and genotyping.

Mobile genetic elements are discrete DNA elements that can move around and copy themselves in a genome. As a ubiquitous component of the genome, mobile elements contribute to both genetic and epigenetic variation. Therefore, it is important to determine the genome-wide distribution of mobile elements. Here we present a targeted high-throughput sequencing protocol called Mobile Element Scanning (ME-Scan) for genome-wide mobile element detection. We will describe oligonucleotides design, sequencing library construction, and computational analysis for the ME-Scan protocol.

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July 7, 2019

Discovery of microbial natural products by activation of silent biosynthetic gene clusters.

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities and a wide variety of applications in medicine and agriculture, such as the treatment of infectious diseases and cancer, and the prevention of crop damage. Genomics has revealed that many microorganisms have far greater potential to produce specialized metabolites than was thought from classic bioactivity screens; however, realizing this potential has been hampered by the fact that many specialized metabolite biosynthetic gene clusters (BGCs) are not expressed in laboratory cultures. In this Review, we discuss the strategies that have been developed in bacteria and fungi to identify and induce the expression of such silent BGCs, and we briefly summarize methods for the isolation and structural characterization of their metabolic products.

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July 7, 2019

Paenibacillus larvae-directed bacteriophage HB10c2 and its application in American Foulbrood-affected honey bee larvae.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious honey bee brood bacterial disease. We isolated and characterized P. larvae-directed bacteriophages and developed criteria for safe phage therapy. Whole-genome analysis of a highly lytic virus of the family Siphoviridae (HB10c2) provided a detailed safety profile and uncovered its lysogenic nature and a putative beta-lactamase-like protein. To rate its antagonistic activity against the pathogens targeted and to specify potentially harmful effects on the bee population and the environment, P. larvae genotypes ERIC I to IV, representatives of the bee gut microbiota, and a broad panel of members of the order Bacillales were analyzed for phage HB10c2-induced lysis. Breeding assays with infected bee larvae revealed that the in vitro phage activity observed was not predictive of the real-life scenario and therapeutic efficacy. On the basis of the disclosed P. larvae-bacteriophage coevolution, we discuss the future prospects of AFB phage therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Novel recA-independent horizontal gene transfer in Escherichia coli K-12.

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F’-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10-12 CFU/recipient per hour.

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July 7, 2019

The assembly and characterisation of two structurally distinct cattle MHC class I haplotypes point to the mechanisms driving diversity.

In cattle, there are six classical MHC class I genes that are variably present between different haplotypes. Almost all known haplotypes contain between one and three genes, with an allele of Gene 2 present on the vast majority. However, very little is known about the sequence and therefore structure and evolutionary history of this genomic region. To address this, we have refined the MHC class I region in the Hereford cattle genome assembly and sequenced a complete A14 haplotype from a homozygous Holstein. Comparison of the two haplotypes revealed extensive variation within the MHC class Ia region, but not within the flanking regions, with each gene contained within a conserved 63- to 68-kb sequence block. This variable region appears to have undergone block gene duplication and likely deletion at regular breakpoints, suggestive of a site-specific mechanism. Phylogenetic analysis using complete gene sequences provided evidence of allelic diversification via gene conversion, with breakpoints between each of the extracellular domains that were associated with high guanine-cytosine (GC) content. Advancing our knowledge of cattle MHC class I evolution will help inform investigations of cattle genetic diversity and disease resistance.

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July 7, 2019

Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis.

Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ?metA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ?metA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ?metA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb-the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action.

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July 7, 2019

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii.

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.

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July 7, 2019

Acetylcholinesterase 1 in populations of organophosphate-resistant North American strains of the cattle tick, Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus, the cattle fever tick, is a global economic problem to the cattle industry due to direct infestation of cattle and pathogens transmitted during feeding. Cattle fever tick outbreaks continue to occur along the Mexico-US border even though the tick has been eradicated from the USA. The organophosphate (OP) coumaphos targets acetylcholinesterase (AChE) and is the approved acaricide for eradicating cattle fever tick outbreaks. There is evidence for coumaphos resistance developing in cattle ticks in Mexico, and OP-resistant R. microplus ticks were discovered in outbreak populations of Texas in 2005. The molecular basis of coumaphos resistance is not known, and our study was established to gather further information on whether AChE1 is involved in the resistance mechanism. We also sought information on allele diversity in tick populations with different levels of coumaphos resistance. The overarching project goal was to define OP resistance-associated gene mutations such that a DNA-based diagnostic assay could be developed to assist the management of resistance. Three different AChE transcripts have been reported in R. microplus, and supporting genomic and transcriptomic data are available at CattleTickBase. Here, we report the complete R. microplus AChE1 gene ascertained by sequencing a bacterial artificial chromosome clone containing the entire coding region and the flanking 5′ and 3′ regions. We also report AChE1 sequences of larval ticks from R. microplus strains having different sensitivities to OP. To accomplish this, we sequenced a 669-bp region of the AChE1 gene corresponding to a 223 amino acid region of exon 2 to assess alleles in seven strains of R. microplus with varying OP resistance phenotypes. We identified 72 AChE1 sequence variants, 2 of which are strongly associated with OP-resistant phenotypes. Esterase-like sequences from the R. microplus transcriptome RmiTr Version 1.0 were compared to the available sequence databases to identify other transcripts with similarity to AChE1.

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July 7, 2019

Sequencing of plasmids pAMBL1 and pAMBL2 from Pseudomonas aeruginosa reveals a blaVIM-1 amplification causing high-level carbapenem resistance.

Carbapenemases are a major concern for the treatment of infectious diseases caused by Gram-negative bacteria. Although plasmids are responsible for the spread of resistance genes among these pathogens, there is limited information on the nature of the mobile genetic elements carrying carbapenemases in Pseudomonas aeruginosa.We combined data from two different next-generation sequencing platforms, Illumina HiSeq2000 and PacBio RSII, to obtain the complete nucleotide sequences of two blaVIM-1-carrying plasmids (pAMBL1 and pAMBL2) isolated from P. aeruginosa clinical isolates.Plasmid pAMBL1 has 26?440 bp and carries a RepA_C family replication protein. pAMBL1 is similar to plasmids pNOR-2000 and pKLC102 from P. aeruginosa and pAX22 from Achromobacter xylosoxidans, which also carry VIM-type carbapenemases. pAMBL2 is a 24?133 bp plasmid with a replication protein that belongs to the Rep_3 family. It shows a high degree of homology with a fragment of the blaVIM-1-bearing plasmid pPC9 from Pseudomonas putida. Plasmid pAMBL2 carries three copies of the blaVIM-1 cassette in an In70 class 1 integron conferring, unlike pAMBL1, high-level resistance to carbapenems.We present two new plasmids coding for VIM-1 carbapenemase from P. aeruginosa and report that the presence of three copies of blaVIM-1 in pAMBL2 produces high-level resistance to carbapenems.© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre recombinase transgene using a novel, efficient, next-generation DNA sequencing method.

It can be useful to know the transgene insertion site in transgenic mice for a variety of reasons, but determining the insertion site generally is a time consuming, expensive, and laborious task.A simple method is presented to determine transgene insertion sites that combines the enrichment of a sequencing library by polymerase chain reaction (PCR) for sequences containing the transgene, followed by next-generation sequencing of the enriched library. This method was applied to determine the site of integration of the thyroid peroxidase promoter-Cre recombinase mouse transgene that is commonly used to create thyroid-specific gene deletions.The insertion site was found to be between bp 12,372,316 and 12,372,324 on mouse chromosome 9, with the nearest characterized genes being Cntn5 and Jrkl, ~1.5 and 0.9?Mbp from the transgene, respectively. One advantage of knowing a transgene insertion site is that it facilitates distinguishing hemizygous from homozygous transgenic mice. Although this can be accomplished by real-time quantitative PCR, the expected Ct difference is only one cycle, which is challenging to assess accurately. Therefore, the transgene insertion site information was used to develop a 3-primer qualitative PCR assay that readily distinguishes wild type, hemizygous, and homozygous TPO-Cre mice based upon size differences of the wild type and transgenic allele PCR products.Identification of the genomic insertion site of the thyroid peroxidase promoter-Cre mouse transgene should facilitate the use of these mice in studies of thyroid biology.

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July 7, 2019

Retrohoming of a mobile group II intron in human cells suggests how eukaryotes limit group II intron proliferation.

Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

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July 7, 2019

Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis.

As part of a genome sequencing project for Ophiocordyceps sinensis, strain 1229, a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 157,510?bp, one of the largest reported for fungi. Conserved genes including the large and small rRNA subunits, 27 tRNA and 15 protein-coding genes, were identified. In addition, 58 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-Seq validated the expression of most conserved genes and ncORFs. Fifty-two introns (groups I and II) were found within conserved genes, accounting for 68.5% of the genome. Thirty-two homing endonucleases (HEs) with motif patterns LAGLIDADG (21) and GIY-YIG (11) were identified in group I introns. The ncORFs found in group II introns mostly encoded reverse transcriptases (RTs). As in other hypocrealean fungi, gene contents and order were found to be conserved in the mt genome of O. sinensis, but the genome size was enlarged by longer intergenic regions and numerous introns. Intergenic and intronic regions were composed of abundant repetitive sequences usually associated with mobile elements. It is likely that intronic ncORFs, which encode RTs and HEs, may have contributed to the enlarged mt genome of O. sinensis.

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