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July 19, 2019

Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue.

Large variation exists in mitochondrial DNA (mtDNA) not only between but also within individuals. Also in human cancer, tumor-specific mtDNA variation exists. In this work, we describe the comparison of four methods to extract mtDNA as pure as possible from frozen tumor tissue. Also, three state-of-the-art methods for sensitive detection of mtDNA variants were evaluated. The main aim was to develop a procedure to detect low-frequent single-nucleotide mtDNA-specific variants in frozen tumor tissue. We show that of the methods evaluated, DNA extracted from cytosol fractions following exonuclease treatment results in highest mtDNA yield and purity from frozen tumor tissue (270-fold mtDNA enrichment). Next, we demonstrate the sensitivity of detection of low-frequent single-nucleotide mtDNA variants (=1% allele frequency) in breast cancer cell lines MDA-MB-231 and MCF-7 by single-molecule real-time (SMRT) sequencing, UltraSEEK chemistry based mass spectrometry, and digital PCR. We also show de novo detection and allelic phasing of variants by SMRT sequencing. We conclude that our sensitive procedure to detect low-frequent single-nucleotide mtDNA variants from frozen tumor tissue is based on extraction of DNA from cytosol fractions followed by exonuclease treatment to obtain high mtDNA purity, and subsequent SMRT sequencing for (de novo) detection and allelic phasing of variants.

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July 19, 2019

Genomic analysis of hospital plumbing reveals diverse reservoir of bacterial plasmids conferring carbapenem resistance.

The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization ofblaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections withblaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission ofLeclerciasp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings.IMPORTANCECarbapenemase-producing organisms (CPOs) are a global concern because of the morbidity and mortality associated with these resistant Gram-negative bacteria. Horizontal plasmid transfer spreads the resistance mechanism to new bacteria, and understanding the plasmid ecology of the hospital environment can assist in the design of control strategies to prevent nosocomial infections. A 5-year genomic and epidemiological survey was undertaken to study the CPOs in the patient-accessible environment, as well as in the plumbing system removed from the patient. This comprehensive survey revealed a vast, unappreciated reservoir of CPOs in wastewater, which was in contrast to the low positivity rate in both the patient population and the patient-accessible environment. While there were few patient-environmental isolate associations, there were plasmid backbones common to both populations. These results are relevant to all hospitals for which CPO colonization may not yet be defined through extensive surveillance.

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July 19, 2019

Dissecting the causal mechanism of X-linked Dystonia-Parkinsonism by integrating genome and transcriptome assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

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July 19, 2019

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.© 2018, Fallon et al.

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July 19, 2019

Coupling of single molecule, long read sequencing with IMGT/HighV-QUEST analysis expedites identification of SIV gp140-specific antibodies from scFv phage display libraries.

The simian immunodeficiency virus (SIV)/macaque model of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome pathogenesis is critical for furthering our understanding of the role of antibody responses in the prevention of HIV infection, and will only increase in importance as macaque immunoglobulin (IG) gene databases are expanded. We have previously reported the construction of a phage display library from a SIV-infected rhesus macaque (Macaca mulatta) using oligonucleotide primers based on human IG gene sequences. Our previous screening relied on Sanger sequencing, which was inefficient and generated only a few dozen sequences. Here, we re-analyzed this library using single molecule, real-time (SMRT) sequencing on the Pacific Biosciences (PacBio) platform to generate thousands of highly accurate circular consensus sequencing (CCS) reads corresponding to full length single chain fragment variable. CCS data were then analyzed through the international ImMunoGeneTics information system®(IMGT®)/HighV-QUEST (www.imgt.org) to identify variable genes and perform statistical analyses. Overall the library was very diverse, with 2,569 different IMGT clonotypes called for the 5,238 IGHV sequences assigned to an IMGT clonotype. Within the library, SIV-specific antibodies represented a relatively limited number of clones, with only 135 different IMGT clonotypes called from 4,594 IGHV-assigned sequences. Our data did confirm that the IGHV4 and IGHV3 gene usage was the most abundant within the rhesus antibodies screened, and that these genes were even more enriched among SIV gp140-specific antibodies. Although a broad range of VH CDR3 amino acid (AA) lengths was observed in the unpanned library, the vast majority of SIV gp140-specific antibodies demonstrated a more uniform VH CDR3 length (20 AA). This uniformity was far less apparent when VH CDR3 were classified according to their clonotype (range: 9-25 AA), which we believe is more relevant for specific antibody identification. Only 174 IGKV and 588 IGLV clonotypes were identified within the VL sequences associated with SIV gp140-specific VH. Together, these data strongly suggest that the combination of SMRT sequencing with the IMGT/HighV-QUEST querying tool will facilitate and expedite our understanding of polyclonal antibody responses during SIV infection and may serve to rapidly expand the known scope of macaque V genes utilized during these responses.

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July 19, 2019

Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains.

Without knowledge of their genomic sequences, it is impossible to make functional models of the bacteria that make up human and animal microbiota. Unfortunately, the vast majority of publicly available genomes are only working drafts, an incompleteness that causes numerous problems and constitutes a major obstacle to genotypic and phenotypic interpretation. In this work, we began with an example from the class Bacteroidia in the phylum Bacteroidetes, which is preponderant among human orodigestive microbiota. We successfully identify the genetic loci responsible for assembly breaks and misassemblies and demonstrate the importance and usefulness of long-read sequencing and curated reannotation.We showed that the fragmentation in Bacteroidia draft genomes assembled from massively parallel sequencing linearly correlates with genomic repeats of the same or greater size than the reads. We also demonstrated that some of these repeats, especially the long ones, correspond to misassembled loci in three reference Porphyromonas gingivalis genomes marked as circularized (thus complete or finished). We prove that even at modest coverage (30X), long-read resequencing together with PCR contiguity verification (rrn operons and an integrative and conjugative element or ICE) can be used to identify and correct the wrongly combined or assembled regions. Finally, although time-consuming and labor-intensive, consistent manual biocuration of three P. gingivalis strains allowed us to compare and correct the existing genomic annotations, resulting in a more accurate interpretation of the genomic differences among these strains.In this study, we demonstrate the usefulness and importance of long-read sequencing in verifying published genomes (even when complete) and generating assemblies for new bacterial strains/species with high genomic plasticity. We also show that when combined with biological validation processes and diligent biocurated annotation, this strategy helps reduce the propagation of errors in shared databases, thus limiting false conclusions based on incomplete or misleading information.

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July 19, 2019

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity.Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel.Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain’s genetic profile from pathogenic to environmental.

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July 19, 2019

Neofunctionalization of duplicated P450 genes drives the evolution of insecticide resistance in the brown planthopper.

Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3-5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6-8]. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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July 19, 2019

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.

Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~?50,000 high-quality full-length HIV envelope sequences (>?2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.

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July 19, 2019

The Florida manatee (Trichechus manatus latirostris) T cell receptor loci exhibit V subgroup synteny and chain-specific evolution.

The Florida manatee (Trichechus manatus latirostris) has limited diversity in the immunoglobulin heavy chain. We therefore investigated the antigen receptor loci of the other arm of the adaptive immune system: the T cell receptor. Manatees are the first species from Afrotheria, a basal eutherian superorder, to have an in-depth characterization of all T cell receptor loci. By annotating the genome and expressed transcripts, we found that each chain has distinct features that correlates to their individual functions. The genomic organization also plays a role in modulating sequence conservation between species. There were extensive V subgroup synteny blocks in the TRA and TRB loci between T. m. latirostris and human. Increased genomic locus complexity correlated to increased locus synteny. We also identified evidence for a VHD pseudogene for the first time in a eutherian mammal. These findings emphasize the value of including species within this basal eutherian radiation in comparative studies. Copyright © 2018. Published by Elsevier Ltd.

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July 19, 2019

HIV envelope glycoform heterogeneity and localized diversity govern the initiation and maturation of a V2 apex broadly neutralizing antibody lineage.

Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Unexpected diversity in the mobilome of a Pseudomonas aeruginosa strain isolated from a dental unit waterline revealed by SMRT Sequencing.

The Gram-negative bacterium Pseudomonas aeruginosa is found in several habitats, both natural and human-made, and is particularly known for its recurrent presence as a pathogen in the lungs of patients suffering from cystic fibrosis, a genetic disease. Given its clinical importance, several major studies have investigated the genomic adaptation of P. aeruginosa in lungs and its transition as acute infections become chronic. However, our knowledge about the diversity and adaptation of the P. aeruginosa genome to non-clinical environments is still fragmentary, in part due to the lack of accurate reference genomes of strains from the numerous environments colonized by the bacterium. Here, we used PacBio long-read technology to sequence the genome of PPF-1, a strain of P. aeruginosa isolated from a dental unit waterline. Generating this closed genome was an opportunity to investigate genomic features that are difficult to accurately study in a draft genome (contigs state). It was possible to shed light on putative genomic islands, some shared with other reference genomes, new prophages, and the complete content of insertion sequences. In addition, four different group II introns were also found, including two characterized here and not listed in the specialized group II intron database.

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July 19, 2019

The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis.

The occurrence of harmful Microcystis blooms is increasing in frequency in a myriad of freshwater ecosystems. Despite considerable research pertaining to the cause and nature of these blooms, the molecular mechanisms behind the cosmopolitan distribution and phenotypic diversity in Microcystis are still unclear. We compared the patterns and extent of DNA methylation in three strains of Microcystis, PCC 7806SL, NIES-2549 and FACHB-1757, using Single Molecule Real-Time (SMRT) sequencing technology. Intact restriction-modification (R-M) systems were identified from the genomes of these strains, and from two previously sequenced strains of Microcystis, NIES-843 and TAIHU98. A large number of methylation motifs and R-M genes were identified in these strains, which differ substantially among different strains. Of the 35 motifs identified, eighteen had not previously been reported. Strain NIES-843 contains a larger number of total putative methyltransferase genes than have been reported previously from any bacterial genome. Genomic comparisons reveal that methyltransferases (some partial) may have been acquired from the environment through horizontal gene transfer. Copyright © 2018 Elsevier B.V. All rights reserved.

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July 19, 2019

Antigenic variation in the lyme spirochete: Insights into recombinational switching with a suggested role for error-prone repair.

The Lyme disease spirochete, Borrelia burgdorferi, uses antigenic variation as a strategy to evade the host’s acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE, and the importance of sequence homology in determining the location of recombination, despite RecA’s dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5′ end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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July 19, 2019

A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat.

Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.© 2018 John Wiley & Sons Ltd.

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