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July 19, 2019

Germinal center centroblasts transition to a centrocyte phenotype according to a timed program and depend on the dark zone for effective selection.

Germinal center (GC) B cells cycle between the dark zone (DZ) and light zone (LZ) during antibody affinity maturation. Whether this movement is necessary for GC function has not been tested. Here we show that CXCR4-deficient GC B cells, which are restricted to the LZ, are gradually outcompeted by WT cells indicating an essential role for DZ access. Remarkably, the transition between DZ centroblast and LZ centrocyte phenotypes occurred independently of positioning. However, CXCR4-deficient cells carried fewer mutations and were overrepresented in the CD73(+) memory compartment. These findings are consistent with a model where GC B cells change from DZ to LZ phenotype according to a timed cellular program but suggest that spatial separation of DZ cells facilitates more effective rounds of mutation and selection. Finally, we identify a network of DZ CXCL12-expressing reticular cells that likely support DZ functions. Copyright © 2013 Elsevier Inc. All rights reserved.

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July 19, 2019

Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis.

More than 20% of the world’s population is at risk for infection by filarial nematodes and >180 million people worldwide are already infected. Along with infection comes significant morbidity that has a socioeconomic impact. The eight filarial nematodes that infect humans are Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi, of which three have published draft genome sequences. Since all have humans as the definitive host, standard avenues of research that rely on culturing and genetics have often not been possible. Therefore, genome sequencing provides an important window into understanding the biology of these parasites. The need for large amounts of high quality genomic DNA from homozygous, inbred lines; the availability of only short sequence reads from next-generation sequencing platforms at a reasonable expense; and the lack of random large insert libraries has limited our ability to generate high quality genome sequences for these parasites. However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries.Here, we report on efforts to generate a more complete genome assembly for L. loa using genetically heterogeneous DNA isolated from a single clinical sample and sequenced on the Pacific Biosciences platform. To obtain the best assembly, numerous assemblers and sequencing datasets were analyzed, combined, and compared. Quiver-informed trimming of an assembly of only Pacific Biosciences reads by HGAP2 was selected as the final assembly of 96.4 Mbp in 2,250 contigs. This results in ~9% more of the genome in ~85% fewer contigs from ~80% less starting material at a fraction of the cost of previous Roche 454-based sequencing efforts.The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.

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July 19, 2019

Transmission of methicillin-resistant Staphylococcus aureus via deceased donor liver transplantation confirmed by whole genome sequencing.

Donor-derived bacterial infection is a recognized complication of solid organ transplantation (SOT). The present report describes the clinical details and successful outcome in a liver transplant recipient despite transmission of methicillin-resistant Staphylococcus aureus (MRSA) from a deceased donor with MRSA endocarditis and bacteremia. We further describe whole genome sequencing (WGS) and complete de novo assembly of the donor and recipient MRSA isolate genomes, which confirms that both isolates are genetically 100% identical. We propose that similar application of WGS techniques to future investigations of donor bacterial transmission would strengthen the definition of proven bacterial transmission in SOT, particularly in the presence of highly clonal bacteria such as MRSA. WGS will further improve our understanding of the epidemiology of bacterial transmission in SOT and the risk of adverse patient outcomes when it occurs.© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

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July 19, 2019

Going beyond five bases in DNA sequencing.

DNA sequencing has provided a wealth of information about biological systems, but thus far has focused on the four canonical bases, and 5-methylcytosine through comparison of the genomic DNA sequence to a transformed four-base sequence obtained after treatment with bisulfite. However, numerous other chemical modifications to the nucleotides are known to control fundamental life functions, influence virulence of pathogens, and are associated with many diseases. These modifications cannot be accessed with traditional sequencing methods. In this opinion, we highlight several emerging single-molecule sequencing techniques that have the potential to directly detect many types of DNA modifications as an integral part of the sequencing protocol. Copyright © 2012 Elsevier Ltd. All rights reserved.

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July 19, 2019

Long-read, whole-genome shotgun sequence data for five model organisms.

Single molecule, real-time (SMRT) sequencing from Pacific Biosciences is increasingly used in many areas of biological research including de novo genome assembly, structural-variant identification, haplotype phasing, mRNA isoform discovery, and base-modification analyses. High-quality, public datasets of SMRT sequences can spur development of analytic tools that can accommodate unique characteristics of SMRT data (long read lengths, lack of GC or amplification bias, and a random error profile leading to high consensus accuracy). In this paper, we describe eight high-coverage SMRT sequence datasets from five organisms (Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, Arabidopsis thaliana, and Drosophila melanogaster) that have been publicly released to the general scientific community (NCBI Sequence Read Archive ID SRP040522). Data were generated using two sequencing chemistries (P4C2 and P5C3) on the PacBio RS II instrument. The datasets reported here can be used without restriction by the research community to generate whole-genome assemblies, test new algorithms, investigate genome structure and evolution, and identify base modifications in some of the most widely-studied model systems in biological research.

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July 19, 2019

Analysis of the Campylobacter jejuni genome by SMRT DNA Sequencing identifies restriction-modification motifs.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni.

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July 19, 2019

CGGBP1 mitigates cytosine methylation at repetitive DNA sequences.

CGGBP1 is a repetitive DNA-binding transcription regulator with target sites at CpG-rich sequences such as CGG repeats and Alu-SINEs and L1-LINEs. The role of CGGBP1 as a possible mediator of CpG methylation however remains unknown. At CpG-rich sequences cytosine methylation is a major mechanism of transcriptional repression. Concordantly, gene-rich regions typically carry lower levels of CpG methylation than the repetitive elements. It is well known that at interspersed repeats Alu-SINEs and L1-LINEs high levels of CpG methylation constitute a transcriptional silencing and retrotransposon inactivating mechanism.Here, we have studied genome-wide CpG methylation with or without CGGBP1-depletion. By high throughput sequencing of bisulfite-treated genomic DNA we have identified CGGBP1 to be a negative regulator of CpG methylation at repetitive DNA sequences. In addition, we have studied CpG methylation alterations on Alu and L1 retrotransposons in CGGBP1-depleted cells using a novel bisulfite-treatment and high throughput sequencing approach.The results clearly show that CGGBP1 is a possible bidirectional regulator of CpG methylation at Alus, and acts as a repressor of methylation at L1 retrotransposons.

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July 19, 2019

HLA typing for the next generation.

Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.

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July 19, 2019

Complete bypass of restriction systems for major Staphylococcus aureus lineages.

Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages.The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus. Copyright © 2015 Monk 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

The impact of next-generation sequencing technologies on HLA research.

In the past decade, the development of next-generation sequencing (NGS) has paved the way for whole-genome analysis in individuals. Research on the human leukocyte antigen (HLA), an extensively studied molecule involved in immunity, has benefitted from NGS technologies. The HLA region, a 3.6-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, primarily autoimmune diseases. Recently, the HLA region has received much attention because severe adverse effects of various drugs are associated with particular HLA alleles. Owing to the complex nature of the HLA genes, classical direct sequencing methods cannot comprehensively elucidate the genomic makeup of HLA genes. Thus far, several high-throughput HLA-typing methods using NGS have been developed. In HLA research, NGS facilitates complete HLA sequencing and is expected to improve our understanding of the mechanisms through which HLA genes are modulated, including transcription, regulation of gene expression and epigenetics. Most importantly, NGS may also permit the analysis of HLA-omics. In this review, we summarize the impact of NGS on HLA research, with a focus on the potential for clinical applications.

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July 19, 2019

HLA Class-II associated HIV polymorphisms predict escape from CD4+ T Cell responses.

Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-? ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4+ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4+ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4+ epitopes and suggests CD4+ T cells are active participants in driving HIV evolution.

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July 19, 2019

Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes.

Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these ‘hotspot’ sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer.

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July 19, 2019

Emergence of ebola virus escape variants in infected nonhuman primates treated with the MB-003 antibody cocktail.

MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Recurrent methicillin-resistant Staphylococcus aureus cutaneous abscesses and selection of reduced chlorhexidine susceptibility during chlorhexidine use.

We describe the selection of reduced chlorhexidine susceptibility during chlorhexidine use in a patient with two episodes of cutaneous USA300 methicillin-resistant Staphylococcus aureus abscess. The second clinical isolate harbors a novel plasmid that encodes the QacA efflux pump. Greater use of chlorhexidine for disease prevention warrants surveillance for resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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