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July 19, 2019

R331W Missense Mutation of Oncogene YAP1 Is a Germline Risk Allele for Lung Adenocarcinoma With Medical Actionability.

Adenocarcinoma is the most dominant type of lung cancer in never-smoker patients. The risk alleles from genome-wide association studies have small odds ratios and unclear biologic roles. Here we have taken an approach featuring suitable medical actionability to identify alleles with low population frequency but high disease-causing potential.Whole-genome sequencing was performed for a family with an unusually high density of lung adenocarcinoma with available DNA from the affected mother, four affected daughters, and one nonaffected son. Candidate risk alleles were confirmed by matrix-assisted laser desorption ionization time of flight mass spectroscopy. Validation was conducted in an external cohort of 1,135 participants without cancer and 1,312 patients with lung adenocarcinoma. Family follow-ups were performed by genotyping the relatives of the original proband and the relatives of the identified risk-allele carriers. Low-dose computed tomography scans of the chest were evaluated for lung abnormalities.YAP1 R331W missense mutation from the original family was identified and validated in the external controls and the cohort with lung adenocarcinoma. The YAP1 mutant-allele carrier frequency was 1.1% in patients with lung adenocarcinoma compared with 0.18% in controls (P = .0095), yielding an odds ratio (adjusted for age, sex, and smoking status) of 5.9. Among the relatives, YAP1-mutant carriers have overwhelmingly higher frequencies of developing lung adenocarcinoma or ground-glass opacity lung lesions than those who do not carry the mutation (10:0 v 1:7; P < .001). YAP1 mutation was shown to increase the colony formation ability and invasion potential of lung cancer cells.These results implicated YAP1 R331W as an allele predisposed for lung adenocarcinoma with high familial penetrance. Low-dose computed tomography scans may be recommended to this subpopulation, which is at high risk for lung cancer, for personalized prevention and health management. © 2015 by American Society of Clinical Oncology.

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July 19, 2019

Insertion sequence IS26 reorganizes plasmids in clinically isolated multidrug-resistant bacteria by replicative transposition.

Carbapenemase-producing Enterobacteriaceae (CPE), which are resistant to most or all known antibiotics, constitute a global threat to public health. Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, is frequently associated with resistance determinants, but its role remains unclear. We have analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the National Institutes of Health Clinical Center. We used target site duplications and their patterns as guides and found that a large fraction of plasmid reorganizations result from IS26 replicative transpositions, including replicon fusions, DNA inversions, and deletions. Replicative transposition could also be inferred for transposon Tn4401, which harbors the carbapenemase blaKPC gene. Thus, replicative transposition is important in the ongoing reorganization of plasmids carrying multidrug-resistant determinants, an observation that carries substantial clinical and epidemiological implications for understanding how such extreme drug resistance phenotypes evolve.Although IS26 is frequently reported to reside in resistance plasmids of clinical isolates, the characteristic hallmark of transposition, target site duplication (TSD), is generally not observed, raising questions about the mode of transposition for IS26. The previous observation of cointegrate formation during transposition implies that IS26 transposes via a replicative mechanism. The other possible outcome of replicative transposition is DNA inversion or deletion, when transposition occurs intramolecularly, and this would also generate a specific TSD pattern that might also serve as supporting evidence for the transposition mechanism. The numerous examples we present here demonstrate that replicative transposition, used by many mobile elements (including IS26 and Tn4401), is prevalent in the plasmids of clinical isolates and results in significant plasmid reorganization. This study also provides a method to trace the evolution of resistance plasmids based on TSD patterns. Copyright © 2015 He et al.

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July 19, 2019

Genetic stabilization of the drug-resistant PMEN1 Pneumococcus lineage by its distinctive DpnIII restriction-modification system.

The human pathogen Streptococcus pneumoniae (pneumococcus) exhibits a high degree of genomic diversity and plasticity. Isolates with high genomic similarity are grouped into lineages that undergo homologous recombination at variable rates. PMEN1 is a pandemic, multidrug-resistant lineage. Heterologous gene exchange between PMEN1 and non-PMEN1 isolates is directional, with extensive gene transfer from PMEN1 strains and only modest transfer into PMEN1 strains. Restriction-modification (R-M) systems can restrict horizontal gene transfer, yet most pneumococcal strains code for either the DpnI or DpnII R-M system and neither limits homologous recombination. Our comparative genomic analysis revealed that PMEN1 isolates code for DpnIII, a third R-M system syntenic to the other Dpn systems. Characterization of DpnIII demonstrated that the endonuclease cleaves unmethylated double-stranded DNA at the tetramer sequence 5′ GATC 3′, and the cognate methylase is a C5 cytosine-specific DNA methylase. We show that DpnIII decreases the frequency of recombination under in vitro conditions, such that the number of transformants is lower for strains transformed with unmethylated DNA than in those transformed with cognately methylated DNA. Furthermore, we have identified two PMEN1 isolates where the DpnIII endonuclease is disrupted, and phylogenetic work by Croucher and colleagues suggests that these strains have accumulated genomic differences at a higher rate than other PMEN1 strains. We propose that the R-M locus is a major determinant of genetic acquisition; the resident R-M system governs the extent of genome plasticity.Pneumococcus is one of the most important community-acquired bacterial pathogens. Pneumococcal strains can develop resistance to antibiotics and to serotype vaccines by acquiring genes from other strains or species. Thus, genomic plasticity is associated with strain adaptability and pneumococcal success. PMEN1 is a widespread and multidrug-resistant highly pathogenic pneumococcal lineage, which has evolved over the past century and displays a relatively stable genome. In this study, we characterize DpnIII, a restriction-modification (R-M) system that limits recombination. DpnIII is encountered in the PMEN1 lineage, where it replaces other R-M systems that do not decrease plasticity. Our hypothesis is that this genomic region, where different pneumococcal lineages code for variable R-M systems, plays a role in the fine-tuning of the extent of genomic plasticity. It is possible that well-adapted lineages such as PMEN1 have a mechanism to increase genomic stability, rather than foster genomic plasticity. Copyright © 2015 Eutsey et al.

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July 19, 2019

Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing.

Single Molecule, Real-Time (SMRT(®)) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

Characterizing and overriding the structural mechanism of the Quizartinib-resistant FLT3 “gatekeeper” F691L mutation with PLX3397.

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase “gatekeeper” residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position.We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop. Cancer Discov; 5(6); 668-79. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 565. ©2015 American Association for Cancer Research.

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July 19, 2019

Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates.

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1 (g609a), and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.

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July 19, 2019

Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae.

Carbapenem resistant Enterobacteriaceae (CRE) pose an urgent risk to global human health. CRE that are non-susceptible to all commercially available antibiotics threaten to return us to the pre-antibiotic era. Using Single Molecule Real Time (SMRT) sequencing we determined the complete genome of a pandrug-resistant Klebsiella pneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics. The precise location of acquired antibiotic resistance elements, including mobile elements carrying genes for the OXA-181 carbapenemase, were defined. Intriguingly, we identified three chromosomal copies of an ISEcp1-blaOXA-181 mobile element, one of which has disrupted the mgrB regulatory gene, accounting for resistance to colistin. Our findings provide the first description of pandrug-resistant CRE at the genomic level, and reveal the critical role of mobile resistance elements in accelerating the emergence of resistance to other last resort antibiotics.

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July 19, 2019

Lineage-specific methyltransferases define the methylome of the globally disseminated Escherichia coli ST131 clone.

Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone. Copyright © 2015 Forde et al.

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July 19, 2019

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology.

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC’s genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring ‘the methylated-motif-site ratio’ and ‘the methylated-read ratio’, we explored the methylation status of each modified site and sequence-read to obtain the ‘precision methylome’ of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

A method for near full-length amplification and sequencing for six hepatitis C virus genotypes.

Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient.In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90 %, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1-3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies.The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications.

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July 19, 2019

Towards better precision medicine: PacBio single-molecule long reads resolve the interpretation of HIV drug resistant mutation profiles at explicit quasispecies (haplotype) level.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient’s HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only = 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients’ HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.

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July 19, 2019

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis d-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd?=?11-41?nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

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July 19, 2019

Initial assessment of the molecular epidemiology of blaNDM-1 in Colombia.

We report complete genome sequences of fourblaNDM-1-harboring Gram-negative multidrug resistant (MDR) isolates from Colombia. TheblaNDM-1genes were located 193Kb-Inc FIA, 178Kb-Inc A/C2 and 47Kb (unknown Inc type) plasmids. MLST revealed that isolates belong to ST10 (Escherichia coli), ST392 (Klebsiella pneumoniae), and ST322 and ST464 (Acinetobacter baumanniiandA. nosocomialis, respectively). Our analysis identified that the Inc A/C2 plasmid inE. colicontained a novel complex transposon (Tn125and Tn5393with 3 copies ofblaNDM-1) and a recombination “hotspot” for the acquisition of new resistance determinants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation.

Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV) replicates via an RNA intermediate and is error-prone, leading to rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome, generated via splicing (spHBV). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. SpHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from longitudinal samples of a post-liver transplant CHB subject, collected over a 15-year period that included the liver explant. By employing novel bioinformatics methods, this analysis showed a complex dynamics of the viral population across a period of changing treatment regimens. The spHBV detected in the liver explant remained present post-transplantation, along with emergence of a highly diverse novel spHBV population as well as variants with multiple deletions in the preS genes. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occur with known drug resistant mutations, highlight the relevance of using full genome deep sequencing and support the hypothesis that drug resistance involves interactions across the full-length HBV genome.Single-molecule sequencing allowed characterising, in unprecedented detail, the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimes. This analysis also showed rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open source bioinformatics tools are freely available, with the capacity to easily identify potential spliced variants from deep sequencing data. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11?kb), single molecule, real-time sequencing.

The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed AT-richness [~80.6% (A?+?T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12?kb, with 50% of the reads between 15.5 and 50?kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [~90-99% (A?+?T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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