Asset Tag: Barcoding
April 29, 2020 |
Procedure & Checklist — Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplex SMRT Sequencing
April 21, 2020 |
Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles: A GeT-RM Collaborative Project.
Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.Published by Elsevier Inc.
April 21, 2020 |
Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.
Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av. © 2019 John Wiley & Sons Ltd.
April 21, 2020 |
IncC blaKPC-2-positive plasmid characterized from ST648 Escherichia coli.
This study describes the characterization of type 2 IncC plasmids pC-Ec20-KPC and pC-Ec2-KPC, carrying blaKPC-2 gene, from two multiresistant E. coli recovered in the University Hospital of Larissa, in 2018.Escherichia coli, Ec-2Lar and Ec-20Lar, were recovered from rectal swabs from two patients, during the monthly surveillance cultures. Transfer experiments by conjugation were carried out with E. coli recipients. blaKPC-carrying plasmids were characterized by S1 profiling. Isolates were typed by MLST. Whole bacterial genome was sequenced using the Sequel platform.Both E. coli isolates, belonging to ST648, transferred the blaKPC-2 to E. coli A15 laboratory strain by conjugation. Plasmid analysis revealed that the transconjugants harbored blaKPC-positive plasmids of different sizes. Analysis of plasmid sequences showed that, in both isolates, blaKPC-2 gene was carried on type 2 IncC plasmids pC-Ec20-KPC and pC-Ec2-KPC. Both plasmids carried the ARI-B resistance island, which consisted of several resistance genes, intact and truncated copies of several mobile elements, and a 25,571-bp segment harboring coding sequences for an iron transporter. The blaKPC-2 gene was part of the transposon Tn4401a, which was bounded by direct repeats of 5 bp (TCCTT) suggesting its transposition into the IncC plasmids.To our knowledge, this is the first report on complete nucleotide sequences of type 2 IncC plasmids. These findings, which hypothesize the acquisition of KPC-2-encoding transposon Tn4401a by an IncC replicon, indicate the ongoing need for molecular surveillance studies of MDR pathogens. Additionally, they underline the increasing clinical importance of the IncC plasmid family.Copyright © 2019. Published by Elsevier Ltd.
April 21, 2020 |
Complete genome sequences of pooled genomic DNA from 10 marine bacteria using PacBio long-read sequencing.
High-quality, completed genomes are important to understand the functions of marine bacteria. PacBio sequencing technology provides a powerful way to obtain high-quality completed genomes. However individual library production is currently still costly, limiting the utility of the PacBio system for high-throughput genomics. Here we investigate how to generate high-quality genomes from pooled marine bacterial genomes.Pooled genomic DNA from 10 marine bacteria were subjected to a single library production and sequenced with eight SMRT cells on the PacBio RS II sequencing platform. In total, 7.35 Gbp of long-read data was generated, which is equivalent to an approximate 168× average coverage for the input genomes. Genome assembly showed that eight genomes with average nucleotide identities (ANI) lower than 91.4% can be assembled with high-quality and completion using standard assembly algorithms (e.g. HGAP or Canu). A reference-based reads phasing step was developed and incorporated to assemble the complete genomes of the remaining two marine bacteria that had an ANI?>?97% and whose initial assemblies were highly fragmented.Ten complete high-quality genomes of marine bacteria were generated. The findings and developments made here, including the reference-based read phasing approach for the assembly of highly similar genomes, can be used in the future to design strategies to sequence pooled genomes using long-read sequencing.Copyright © 2019. Published by Elsevier B.V.
April 21, 2020 |
The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.
Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4+ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
April 21, 2020 |
Broadly Neutralizing Antibodies Targeting New Sites of Vulnerability in Hepatitis C Virus E1E2.
Increasing evidence indicates that broadly neutralizing antibodies (bNAbs) play an important role in immune-mediated control of hepatitis C virus (HCV) infection, but the relative contribution of neutralizing antibodies targeting antigenic sites across the HCV envelope (E1 and E2) proteins is unclear. Here, we isolated thirteen E1E2-specific monoclonal antibodies (MAbs) from B cells of a single HCV-infected individual who cleared one genotype 1a infection and then became persistently infected with a second genotype 1a strain. These MAbs bound six distinct discontinuous antigenic sites on the E1 protein, the E2 protein, or the E1E2 heterodimer. Three antigenic sites, designated AS108, AS112 (an N-terminal E1 site), and AS146, were distinct from previously described antigenic regions (ARs) 1 to 5 and E1 sites. Antibodies targeting four sites (AR3, AR4-5, AS108, and AS146) were broadly neutralizing. These MAbs also displayed distinct patterns of relative neutralizing potency (i.e., neutralization profiles) across a panel of diverse HCV strains, which led to complementary neutralizing breadth when they were tested in combination. Overall, this study demonstrates that HCV bNAb epitopes are not restricted to previously described antigenic sites, expanding the number of sites that could be targeted for vaccine development.IMPORTANCE Worldwide, more than 70 million people are infected with hepatitis C virus (HCV), which is a leading cause of hepatocellular carcinoma and liver transplantation. Despite the development of potent direct acting antivirals (DAAs) for HCV treatment, a vaccine is urgently needed due to the high cost of treatment and the possibility of reinfection after cure. Induction of multiple broadly neutralizing antibodies (bNAbs) that target distinct epitopes on the HCV envelope proteins is one approach to vaccine development. However, antigenic sites targeted by bNAbs in individuals with spontaneous control of HCV have not been fully defined. In this study, we characterize 13 monoclonal antibodies (MAbs) from a single person who cleared an HCV infection without treatment, and we identify 3 new sites targeted by neutralizing antibodies. The sites targeted by these MAbs could inform HCV vaccine development. Copyright © 2019 American Society for Microbiology.
April 21, 2020 |
Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.
The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting non-ribosomal peptides (NRPS) and Type I polyketides (PKS). NRPS-encoding genes were more widespread across the Antarctic, whereas PKS genes were only recoverable from a handful of sites. Many recovered sequences were deemed novel due to their low amino acid sequence similarity to known protein sequences, particularly throughout the east Antarctic sites. Phylogenetic analysis revealed that a high proportion were most similar to antifungal and biosurfactant-type clusters. Multivariate analysis showed that soil fertility factors of carbon, nitrogen and moisture displayed significant negative relationships with natural product gene richness. Our combined results suggest that secondary metabolite production is likely to play an important physiological component of survival for microorganisms inhabiting arid, nutrient-starved soils. © FEMS 2019.
April 21, 2020 |
CD8 T cells targeting adapted epitopes in chronic HIV infection promote dendritic cell maturation and CD4 T cell trans-infection.
HIV-1 frequently escapes from CD8 T cell responses via HLA-I restricted adaptation, leading to the accumulation of adapted epitopes (AE). We previously demonstrated that AE compromise CD8 T cell responses during acute infection and are associated with poor clinical outcomes. Here, we examined the impact of AE on CD8 T cell responses and their biological relevance in chronic HIV infection (CHI). In contrast to acute infection, the majority of AE are immunogenic in CHI. Longitudinal analyses from acute to CHI showed an increased frequency and magnitude of AE-specific IFN? responses compared to NAE-specific ones. These AE-specific CD8 T cells also were more cytotoxic to CD4 T cells. In addition, AE-specific CD8 T cells expressed lower levels of PD1 and CD57, as well as higher levels of CD28, suggesting a more activated and less exhausted phenotype. During CHI, viral sequencing identified AE-encoding strains as the dominant quasispecies. Despite increased CD4 T cell cytotoxicity, CD8 T cells responding to AE promoted dendritic cell (DC) maturation and CD4 T cell trans-infection perhaps explaining why AE are predominant in CHI. Taken together, our data suggests that the emergence of AE-specific CD8 T cell responses in CHI confers a selective advantage to the virus by promoting DC-mediated CD4 T cell trans-infection.
April 21, 2020 |
Targeted Long-Read RNA Sequencing Demonstrates Transcriptional Diversity Driven by Splice-Site Variation in MYBPC3.
To date, clinical sequencing has focused on genomic DNA using targeted panels and exome sequencing. Sequencing of a large hypertrophic cardiomyopathy (HCM) cohort revealed that positive identification of a disease-associated variant was returned in only 32% of patients, with an additional 15% receiving inconclusive results. When genome sequencing fails to reveal causative variants, the transcriptome may provide additional diagnostic clarity. A recent study examining patients with genetically undiagnosed muscle disorders found that RNA sequencing, when used as a complement to exome and whole genome sequencing, had an overall diagnosis rate of 35%.
April 21, 2020 |
µLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome.
Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the µLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/µl for 50 kb fragments and an analytical time of 50 min. Then, µLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with µLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with µLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
April 21, 2020 |
Antigenic variation in the Lyme spirochete: detailed functional assessment of recombinational switching at vlsE in the JD1 strain of Borrelia burgdorferi.
Borrelia burgdorferi is a causative agent of Lyme disease and establishes long-term infection in mammalian hosts. Persistence is promoted by the VlsE antigenic variation system, which generates combinatorial diversity of VlsE through unidirectional, segmental gene conversion from an array of silent cassettes. Here we explore the variants generated by the vls system of strain JD1, which has divergent sequence and structural elements from the type strain B31, the only B. burgdorferi strain in which recombinational switching at vlsE has been studied in detail. We first completed the sequencing of the vls region in JD1, uncovering a previously unreported 114 bp inverted repeat sequence upstream of vlsE. A five-week infection of WT and SCID mice was used for PacBio long read sequencing along with our recently developed VAST pipeline to analyze recombinational switching at vlsE from 40,000 sequences comprising 226,000 inferred recombination events. We show that antigenic variation in B31 and JD1 is highly similar, despite the lack of 17 bp direct repeats in JD1, a somewhat different arrangement of the silent cassettes, divergent inverted repeat sequences and general divergence in the vls sequences. We also present data that strongly suggest that dimerization is required for in vivo functionality of VlsE. © 2018 John Wiley & Sons Ltd.
April 21, 2020 |
SMRT sequencing revealed the diversity and characteristics of defective interfering RNAs in influenza A (H7N9) virus infection.
Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.
April 21, 2020 |
a-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY.
Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent a-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.