Multiple Variable Regions Research Articles

Updating Urinary Microbiome Analyses to Enhance Biologic Interpretation.

ObjectiveAn approach for assessing the urinary microbiome is 16S rRNA gene sequencing, where analysis methods are rapidly evolving. This re-analysis of an existing dataset aimed to determine whether updated bioinformatic and statistical techniques affect clinical inferences.MethodsA prior study compared the urinary microbiome in 123 women with mixed urinary incontinence (MUI) and 84 controls. We obtained unprocessed sequencing data from multiple variable regions, processed operational taxonomic unit (OTU) tables from the original analysis, and de-identified clinical data. We re-processed sequencing data with DADA2 to generate amplicon sequence variant (ASV) tables. Taxa from ASV tables were compared to the original OTU tables; taxa from different variable regions after updated processing were also compared. Bayesian graphical compositional regression (BGCR) was used to test for associations between microbial compositions and clinical phenotypes (e.g., MUI versus control) while adjusting for clinical covariates. Several techniques were used to cluster samples into microbial communities. Multivariable regression was used to test for associations between microbial communities and MUI, again while adjusting for potentially confounding variables.ResultsOf taxa identified through updated bioinformatic processing, only 40% were identified originally, though taxa identified through both methods represented >99% of the sequencing data in terms of relative abundance. Different 16S rRNA gene regions resulted in different recovered taxa. With BGCR analysis, there was a low (33.7%) probability of an association between overall microbial compositions and clinical phenotype. However, when microbial data are clustered into bacterial communities, we confirmed that bacterial communities are associated with MUI. Contrary to the originally published analysis, we did not identify different associations by age group, which may be due to the incorporation of different covariates in statistical models.ConclusionsUpdated bioinformatic processing techniques recover different taxa compared to earlier techniques, though most of these differences exist in low abundance taxa that occupy a small proportion of the overall microbiome. While overall microbial compositions are not associated with MUI, we confirmed associations between certain communities of bacteria and MUI. Incorporation of several covariates that are associated with the urinary microbiome improved inferences when assessing for associations between bacterial communities and MUI in multivariable models.

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method.

Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

Sequence variation and immunogenicity of the Mycoplasma genitalium MgpB and MgpC adherence proteins during persistent infection of men with non-gonococcal urethritis.

Mycoplasma genitalium is a sexually transmitted bacterial pathogen that infects men and women. Antigenic variation of MgpB and MgpC, the immunodominant adherence proteins of M. genitalium, is thought to contribute to immune evasion and chronic infection. We investigated the evolution of mgpB and mgpC sequences in men with non-gonococcal urethritis persistently infected with M. genitalium, including two men with anti-M. genitalium antibodies at enrollment and two that developed antibodies during follow-up. Each of the four patients was persistently infected with a different strain type and each patient produced antibodies targeting MgpB and MgpC. Amino acid sequence evolution in the variable regions of MgpB and MgpC occurred in all four patients with changes observed in single and multiple variable regions over time. Using the available crystal structure of MgpC of the G37 type strain we found that predicted conformational B cell epitopes localize predominantly to the variable region of MgpC, amino acids that changed during patient infection lie in these epitopes, and variant amino acids are in close proximity to the conserved sialic acid binding pocket. These findings support the hypothesis that sequence variation functions to avoid specific antibodies thereby contributing to persistence in the genital tract.

The Assessment of Bacterial and Fungal Biodiversity in Korean Dermatomycoses Patients: A Pilot Study

Background: Dermatophytes are the most common cause of fungal infections worldwide, having an estimated prevalence of 20% by the World Health Organization. Recent advances of sequencing and annotation of fungi explain the biodiversity evaluation and genomic identification of dermatophytes. Objective: This study's objective is to assess the mycobiome and microbiome in different dermatomycoses and identify their pathogenic organisms using fungal genomic analyses. Besides, we wanted to see the topographical biodiversity in dermatomycoses. Methods: Patients with dermatophyte infections confirmed by positive potassium peroxide examination were enrolled. Bacterial and fungal DNA were extracted from the skin sample. The internal transcribed spacer regions of rRNA gene and multiple variable regions in 16S rRNA gene were assessed to determine the fungal and bacterial community compositions. Results: We profiled both bacterial and fungal communities in the fungal-infected skin. Dermatophyte infection resulted in modified bacterial microbiome. The alpha diversity showed that the bacterial diversity was relatively low in tinea cruris patients compared to other dermatomycoses patients. Nonetheless, fungal diversity was not impacted by the body site but by the causative fungal organisms. Tinea pedis, tinea corporis, and tinea cruris due to Trichophyton species were similar as seen in principle component analysis. Alpha diversity demonstrates the tinea corporis on the leg had the most diverse fungal mycobiome and the scalp is the least diverse area. Conclusion: Sequencing and annotation of dermatophyte genomes of superficial skin infections will give sources that will help explain the virulence mechanisms of the dermatophyte. Understanding the pathogenesis of dermatophyte infections can help in generating effective therapeutics in the future.

Abstract 1484: Microbial sequencing using a single-pool target enrichment of multiple variable regions of the 16S rRNA gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and antimicrobial resistance genes

Abstract The human microbiome has been studied in the context of a number of diseases and has recently been gaining momentum in the field of cancer research. The composition of the gut microbiome, for example, has been shown to affect cancer progression and may act as both a biomarker and therapeutic target. Metagenomic shotgun sequencing has played a large role in characterizing the composition of microbial communities. However, direct sequencing of multiple complex samples in parallel has relatively low sensitivity and struggles to accurately characterize the microbiome. Alternatively, targeted PCR and sequencing can be used to simultaneously enrich for and identify multiple microbial targets of interest in parallel. Here, we present a single-pool multiplex PCR assay to enrich for multiple variable regions of the 16S ribosomal RNA (rRNA) gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and 35 antibiotic resistance genes from multiple bacteria species listed as threats by the CDC. Our two-hour protocol generates target-enriched libraries from genomic DNA (gDNA) that are compatible with Illumina sequencing platforms. We validated our protocol using gDNA from mock microbial communities comprising 20 bacterial strains mixed at varied proportions. The specificity, sensitivity, and quantification of our assay was also evaluated using clinical isolates and extracts from swine manure. We found that our single-tube, target-enriched library workflow accurately and reproducibly identified genomic content from reference strains with as few as six starting genomic copies mixed in complex samples. Further, enrichment of seven variable (V) regions of the 16S rRNA gene facilitated the detection of greater diversity and improved classification of microbial communities compared to the conventionally used primer set, which only targets the V3-V4 regions. Simultaneous analysis of both the 16S rRNA gene and antibiotic resistance genes provided greater differentiation of bacterial pathogens and greater characterization of the overall structure of the community’s functional repertoire. This study highlights the power of targeted enrichment via single-pool amplification for sequencing and characterizing microbial communities, including those that have important consequences for human health. Citation Format: Kayla Peck, Robert Stedtfeld, Jordan RoseFigura, Brett Reed, Drew McUsic, Jon Irish, Brett Etchebarne, Timothy Johnson, Laurie Kurihara, Vladimir Makarov. Microbial sequencing using a single-pool target enrichment of multiple variable regions of the 16S rRNA gene, the nuclear ribosomal internal transcribed spacer (ITS) region, and antimicrobial resistance genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1484.

A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities

Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches.We developed a method that identifies and quantifies members of bacterial communities through simultaneous analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines high-throughput microfluidics for PCR amplification, short read DNA sequencing, and a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON) for optimizing taxonomic assignment. MVRSION performance was compared to single variable region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains using existing software (QIIME), and the results of community profiling by shotgun sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized with a defined, phylogenetically diverse consortium of human gut bacterial strains. Positive predictive values for MVRSION ranged from 65%–91% versus 44%–61% for single region QIIME analyses (p < .01, p < .001), while the abundance estimate r2 for MVRSION compared to COPRO-Seq was 0.77 vs. 0.46 and 0.45 for V3-QIIME and V4-QIIME, respectively.MVRSION represents a generally applicable tool for taxonomic classification that is superior to single-region 16S rRNA methods, resource efficient, highly scalable for assessing the microbial composition of up to thousands of samples concurrently, with multiple applications ranging from whole community profiling to targeted tracking of organisms of interest in diverse habitats as a function of specified variables/perturbations.

Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing

Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when applied to patient samples with limited volumes. Here, we demonstrate an optimized molecular inversion probe (MIP) assay targeting multiple variable regions within the 16S ribosomal gene for the identification of biothreat and ESKAPE pathogens in a process that significantly reduces complexity, labor, and processing time. Probes targeting the Klebsiella pneumoniae carbapenemase (KPC) antibiotic resistance (AR) gene were also included to demonstrate the ability to concurrently identify etiologic agent and ascertain valuable secondary genetic information. Our assay captured gene sequences in 100% of mock clinical samples prepared from flagged positive blood culture bottles. Using a simplified processing and adjudication method for mapped sequencing reads, genus and species level concordance was 100% and 80%, respectively. In addition, sensitivity and specificity for KPC gene detection was 100%. Our MIP assay produced sequenceable amplicons for the identification of etiologic agents and the detection of AR genes directly from blood culture bottles in a simplified single tube assay.

Comparison of Brucella canis genomes isolated from different countries shows multiple variable regions

Brucella canis is a pathogenic bacterium for dogs and its zoonotic potential has been increasing in recent years. In this study, we report the sequencing, annotation and analysis of the genome of Brucella canis strain Oliveri isolated from a dog in a breeding kennel in Medellín, Colombia, South America.Whole genome shotgun sequencing was carried out using the ROCHE 454 GS FLX Titanium technology at the National Center for Genomic Sequencing—CNSG in Medellin, Colombia. The assembly procedure was performed using Newbler v2.6. In the genome annotation process, each contig was analyzed independently using as reference Brucella suis ATCC 1330 chromosomes.This new genome could be useful for the development of diagnostic tools and for vaccines search as well, in order to reduce the health impact of this infection in both, dogs and humans. The sequence was deposited in EMBL-EBI with accession numbers HG803175 and HG803176 for chromosomes 1 and 2, respectively.

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

Analysis of the Immunoglobulin Light Chain Genes in Zebra Finch: Evolutionary Implications

All jawed vertebrates produce immunoglobulins (IGs) as a defense mechanism against pathogens. Typically, IGs are composed of two identical heavy chains (IGH) and two identical light chains (IGL). Most tetrapod species encode more than one isotype of light chains. Chicken is the only representative of birds for which genomic information is currently available and is an exception to the above rule because it encodes only a single IGL isotype (i.e., lambda). Here, we show that the genome of zebra finch, another bird species, encodes a single IGL isotype, that is, lambda, like the chicken. These results strongly suggest that the second isotype (i.e., kappa) present in both reptiles and mammals was lost in a very early stage of bird evolution. Furthermore, we show that both chicken and zebra finch contain a single set of functional variable, joining, and constant region genes and multiple variable region pseudogenes. The latter finding suggests that this type of genomic organization was already present in the common ancestor of these bird species and remained unchanged over a long evolutionary time. This conservation is in contrast with the high levels of variation observed in the mammalian IGL loci. The presence of a single functional variable region gene followed by multiple variable pseudogenes in zebra finch suggest that this species may be generating antibody diversity by a gene conversion-like mechanism like the chicken.

The InDeVal insertion/deletion evaluation tool: a program for finding target regions in DNA sequences and for aiding in sequence comparison

BackgroundThe program InDeVal was originally developed to help researchers find known regions of insertion/deletion activity (with the exception of isolated single-base indels) in newly determined Poaceae trnL-F sequences and compare them with 533 previously determined sequences. It is supplied with input files designed for this purpose. More broadly, the program is applicable for finding specific target regions (referred to as "variable regions") in DNA sequence. A variable region is any specific sequence fragment of interest, such as an indel region, a codon or codons, or sequence coding for a particular RNA secondary structure.ResultsInDeVal input is DNA sequence and a template file (sequence flanking each variable region). Additional files contain the variable regions and user-defined messages about the sequence found within them (e.g., taxa sharing each of the different indel patterns).Variable regions are found by determining the position of flanking sequence (referred to as "conserved regions") using the LPAM (Length-Preserving Alignment Method) algorithm. This algorithm was designed for InDeVal and is described here for the first time.InDeVal output is an interactive display of the analyzed sequence, broken into user-defined units. Once the user is satisfied with the organization of the display, the information can be exported to an annotated text file.ConclusionsInDeVal can find multiple variable regions simultaneously (28 indel regions in the Poaceae trnL-F files) and display user-selected messages specific to the sequence variants found. InDeVal output is designed to facilitate comparison between the analyzed sequence and previously evaluated sequence. The program's sensitivity to different levels of nucleotide and/or length variation in conserved regions can be adjusted. InDeVal is currently available for Windows in Additional file 1 or from .

Neutralization epitopes of the HIV-1 primary isolate DH012

To study HIV-1 primary isolate neutralization, we have used DH012 as a model to study the immunogenicity of several DH012 immunogens and determine the potential neutralization epitopes in the virus envelope glycoprotein. Previously, we identified that DH012 infected animals mount potent neutralizing activity against a conformational epitope (CEV) that involves multiple variable regions. In this study, we show that the conformational epitope can be reconstituted with one gp120 recombinant fragment containing sequences from the V1/V2 loop and the bridging sheet of gp120 and a V3 peptide. In contrast to DH012 infection, we previously demonstrated that animals immunized with DH012 gp120 induced potent neutralizing antibodies directed at the V3 domain of gp120. In this study, a second neutralizing activity against the V1/V2 region of gp120 was identified from the same guinea pig sera. In summary, several neutralization epitopes are identified on DH012, including the CEV, V1/V2, V3, 17b, IgG1b12, and 2G12 epitopes. Infectious DH012 virus carrying oligomeric envelope appears to raise primarily neutralizing antibodies that recognize a discontinuous conformationally dependent epitope whereas the monomeric gp120 induces antibodies that are primarily directed at epitopes in the V3 and V1/V2 domains. The DH012 neutralizing epitopes, such as V1/V2 and V3, are either cryptic or poorly immunogenic in chimpanzees. However, immunogens, such as gp120, could be designed to induce neutralizing activity against epitopes that are poorly immunogenic, such as V1/V2 of DH012, in the native envelope glycoproteins.

Interspecific polymorphism at non-coding regions of chloroplast, mitochondrial DNA and rRNA IGS region in Elymus species.

Several published universal primers for amplification of non-coding regions of chloroplast, mitochondrial and ribosomal (rRNA) IGS region were tested whether they can amplify respective regions in Elymus species. PCR-RFLP analysis of the chloroplast, mitochondral DNA, and rRNA IGS region of the genus Elymus was used to determine if the method could be employed to detect inter-specific variation in this genus. Published universal primers for amplification of trnK [tRNA-Lys (UUU) exon 1]-trnK [tRNA-Lys (UUU) exon2], and mitochondrial nad1 exon B-nadl exon C intron successfully amplified the respective regions in Elymus species. However, the primers for amplification of chloroplast trnD-trnT intron and rRNA IGS failed to amplify the respective region in Elymus species. New primer pairs were designed and successfully amplified the cpDNA trnD-trnT intron and rRNA IGS region in Elymus species. The amplification products were digested with seven restriction enzymes. The results showed that the investigated regions of chloroplast and mitochondrial genomes are variable in most of the tested taxa and contain multiple variable regions. These regions should serve as useful molecular markers in phylogenetic studies of closely related species, at least at the interspecific level in Elymus. It is likely that further studies, including larger sample sizes, more regions of these genomes and/or more powerful methods for the detection of cpDNA and mt DNA variation will reveal additional variation for this genus. Highly inter- and intra-specific polymorphisms for rRNA IGS region were detected, suggesting the IGS will be a useful molecular marker for population studies of Elymus species.

PCR-RFLP analysis of cpDNA in the genus Abies

We used PCR-RFLP analysis of the chloroplast DNA of the genus Abies (family Pinaceae), to determine if the method could be employed to detect inter-specific variation in this genus and to study how the variation was distributed in different regions of the genome. Ten different chloroplast DNA regions, consisting of coding and non-coding DNA sequences, were amplified with specific primers in ten different Abies taxa. The amplification products were digested with several restriction enzymes. The results showed that the chloroplast genome is highly variable in most of the investigated taxa and contains multiple variable regions that appear to be distributed throughout the whole genome. Species-diagnostic markers were found for four of the ten investigated species. Unexpectedly, intra-specific variation was also detected in four species. It is likely that further studies, including larger sample sizes and/or more powerful methods for the detection of chloroplast DNA variation, will reveal additional variation for this genus.

Infection of chimpanzee peripheral blood mononuclear cells by human immunodeficiency virus type 1 requires cooperative interaction between multiple variable regions of gp120.

We have recently reported the isolation and molecular cloning of a human immunodeficiency virus type 1 isolate (HIV-1 DH125) that exhibits rapid replication kinetics and marked cytopathicity in both human and chimpanzee peripheral blood mononuclear cells (PBMC). To identify the viral determinants responsible for infectivity of chimpanzee PBMC, chimeric viruses containing the following components were constructed: (i) the entire envelope gene; (ii) gp120 sequences; (iii) gp41 sequences; and (iv) individual or various combinations of the gp120 variable regions of HIV-1 DH125 inserted into the backbone of another HIV-1 isolate (HIV-1 AD8), which is unable to infect chimpanzee PBMC. Analyses of virus replication kinetics in human and chimpanzee PBMC revealed that gp120 contains determinants which confer infectivity for chimpanzee PBMC and that the capacity to establish such an infection requires the cooperative interaction between multiple variable regions of the HIV-1 DH125 gp120.

Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions.

Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions.

Subclustering of Human Immunoglobulin Kappa Light Chain Variable Region Genes

The human immunoglobulin kappa light chain (IgK) locus includes multiple variable region gene segments (Vk) that can be divided into four subgroups. Oligonucleotide primers were designed to amplify specifically gene segments of the VkI, VkII, and Vk III subgroups using the polymerase chain reaction (PCR). Product sequences were subcloned, sequenced, and compared. Phylogenetic analyses of sequences within each subgroup indicate that some subgroups can be subdivided further into "sub-subgroups." The history of Vk segment duplications apparently includes at least two separate periods, the first giving rise to the subgroups and the second generating further complexity within each subgroup. Duplications of large pieces of DNA (demonstrated by others through pulsed-field gel electrophoresis) also played a role. Rates of synonymous and nonsynonymous base changes between pairs of sequences suggest that natural selection has played a major role in the evolution of the Vk variable gene segments, leading to sequence conservation in some regions and to increased diversity in others.

Influence of T cell receptor V alpha expression on Mlsa superantigen-specific T cell responses.

Recognition of conventional foreign antigen by T cells is determined by the expression of multiple variable regions of both alpha and beta chains of the T cell receptor (TCR) alpha/beta heterodimer. In contrast, there exists a class of antigens that appears to interact with the TCR alpha/beta heterodimer through the variable region on the beta chain (V beta), independent of other TCR components, a property that has led to their designation as superantigens. The goal of the present study was to analyze V alpha use in V beta 6+ T cells responsive to the superantigen, Mlaa. Results indicate that while deletion of T cells expressing V beta 6 in Mlsa-expressing mice is essentially complete and therefore appears to occur regardless of V alpha usage, in vitro Mlsa stimulation of T cells from Mlsa-negative mice results in significant skewing of V alpha use among responding V beta 6+ T cells. This indicates that V alpha expression influences recognition of the superantigen, Mlsa by mature peripheral T cells.