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To date, a significant amount of work has been performed on the human microbiome to evaluate its composition and influence on physiology; this research has led to additional studies on microbiomes localized at specific sites of the human body (e.g., skin, oral, vaginal). Given that fungi are ubiquitous and live in symbiosis with the human body, researchers are now actively looking into the role of the mycobiome in human health and disease. Recent advancements in sequencing technologies have enabled the community profiling of fungi; however, the complexities associated with metagenomics sequencing analyses have posed significant challenges toward standardization. To address this need, ATCC has developed genomic DNA and whole cell mock microbial communities comprising ten medically relevant fungal species mixed in even proportions. In this proof-of-concept study, we demonstrate the use these standards in evaluating DNA extraction and sequencing methods for mycobiome analysis.

A predominant limitation in microbiome research is the lack of appropriate and relevant standards to control the technical biases introduced throughout the metagenomics workflow. To address this, ATCC has developed a set of genomic DNA and whole cell mock microbial communities from fully sequenced and characterized ATCC strains that represent species found in the oral, skin, gut, or vaginal microbiome. Here, we demonstrate the utility of these standards as reference materials for 16S and shotgun analyses performed on long-read sequencing platforms. This proof-of-concept analysis demonstrate that ATCC® Microbiome Standards are platform agnostic and can be used for the development and optimization of assays performed on both short-read and long-read sequencing platforms.

Microbial Genome Sequencing and Assembly

Existing Challenges and the Need for Authentic Reference Genomes

6/20/2019 — 6/24/2019

The advancement and accessibility of next-generation sequencing (NGS) technologies have rapidly transformed microbiological research by providing the ability to analyze and profile microbial communities via metagenomics analyses. These sequencing-based applications have relied on the availability of fully assembled reference genomes for bioinformatics analyses, particularly for variant calling in diagnostic and clinical microbiology. However, despite the availability of existing genome sequences in public databases, the quality, completeness, authenticity, accuracy, and traceability of genomic data is inadequate; the lack of standards for genome quality exacerbates these underlying problems. To address this, ATCC has implemented a robust NGS and genome assembly workflow to advance authentication of bacterial strains in the ATCC collection.

The Bacillus cereus Group (BcG) is a group of closely related species that are important in health (e.g., B. anthracis and B. cereus) and biotechnology (e.g., B. thuringiensis). Recently, many new species were added to the BcG, bringing the current total to 18 species. With this recent expansion of the BcG, it is useful to revisit the species classification of existing BcG strains to determine whether their species assignments require realignment with the most current taxonomy of the group. Here, we evaluated the genome-to-genome distances (GGDs) between the whole-genome sequences of 35 strains and that of the 18 BcG type strains present in GenBank by using the Genome-to-Genome Distance Calculator (GGDC). The phylogenomic analysis described here illustrates the importance of reexamining the identity of existing strains via the most recent tools and taxonomic information. Particularly with items deposited decades before our modern understanding of genotypic characterization, such phylogenomic reexaminations enable the taxonomic reassessment of strains to ensure their accurate alignment with the most current taxonomy.

Mycobacterium tuberculosis (Mtb), a causative agent for tuberculosis (TB), remains one of the most challenging pathogens to control. Mtb infects nearly a quarter of the world’s population and sinisterly synergizes with HIV to claim the lives of around 1/3 of all AIDS patients. While the WHO has considered this epidemic a global emergency since 1994, TB control has been hampered by lack of protective vaccines and a rapid, effective diagnostic tool. Only in recent years has TB antibody development offered new potential to control TB infections. The humoral antibody functions of TB immunity have been discovered and distinguishable antibody patterns in active/chronic stages of TB have been uncovered. Thus, knowledge of the paratope sequences of Mtb antibodies enables engineering diagnostic and therapeutic tools. Currently, few validated TB antibody sequences are available. Here, we identified the sequence of functional variable immunoglobulins (IgVs) expressed in 14 hybridomas encoding antibodies to Mtb targets with potential therapeutic/diagnostic value: (1) Mpt64, a mycobacterial diagnostic peptide; (2) Ag85 complex, the most immunogenic Mtb protein to date; (3) the Mtb bacterial surface components (A) glycolipid LAM, (B) lipoprotein LprG, and (C) HBHA, an epithelial cell adhesion factor; (4) the Mtb enzymes (A) Superoxide Dismutase SodA and (B) Catalase KatG, crucial for Mtb survival within the hostile macrophage phagosome; (5) the Mtb regulatory factors PhoS1/PstS1, factors within the ABC transporter system; and (6) the Mtb Heat Shock Proteins HspX, DnaK, and GroES. We isolated the paratope-determining CDR 1-3 regions of the heavy and light chains of these IgVs using a 5’RACE-PCR amplification from the cDNA of each hybridoma via an isotype (gene)-specific primer (GSP) for each of the light chains of Igk/Igl_1, 2, and 3 and heavy chains of IgG1, IgG2a, IgG3, and IgM. Using an Illumina® NGS-MiSeq™, 2X150bp, pair-wise sequencing platform, we generated 28 IgV libraries. Most libraries contained sufficient reads and coverage for de novo assembly of Ig chains via a bioinformatics algorithm workflow for analysis of Ig sequences. Thirty-three (33) putative TB IgV sequences were identified. Validation of their antigen-binding capability via recombinant antibody techniques is in progress.

Most commonly known for their probiotic properties, Bifidobacteria are gram-positive, rod-shaped, anaerobic bacteria often found in the digestive tracts of various mammals and insects. Belonging to the same family as Bifidobacterium, Gardnerella comprises a single species (G. vaginalis) implicated in bacterial vaginosis, whose taxonomic status has often been disputed. Various phylogenetic trees of the Bifidobacteriaceae family place G. vaginalis centrally within the Bifidobacterium genus on the basis of 16S rRNA sequences. Additionally, the discrimination between Gardnerella and Bifidobacterium has proven difficult in the laboratory. In this study, we aim to elucidate the taxonomic position of G. vaginalis and revise the classification of the Bifidobacterium genus through whole-genome sequencing (WGS) of 62 type strains of Bifidobacterium and the type strain of G. vaginalis

Comprising 19 species/subspecies, Yersinia are Gram-negative coccobacilli implicated in a variety of human and zoonotic diseases. Several species of Yersinia share high genomic similarity with each other, and the ability to discern these species is vital—particularly for Y. pestis, the causative agent of plague, whose genomic composition is closely related to Y. pseudotuberculosis. In this study, we aim to revisit the taxonomy of Yersinia through whole-genome sequencing (WGS) of the type strains and confirm their taxonomic assignment. Whole-genome distances and phylogenomic analyses confirmed the current taxonomy of 17 species/subspecies. Unsurprisingly, four species that showed a greater degree of relatedness are Y. pestis, Y. pseudotuberculosis, Y. similis, and Y. wautersii, which constitute the Y. pseudotuberculosis complex. Recent research using multilocus sequence analysis identified Y. wautersii as a novel member species of the Y. pseudotuberculosis complex. However, based on whole-genome distances, our data shows enough similarity between Y. wautersii and Y. pseudotuberculosis to be considered the same species but different subspecies. Phylogenomic trees, which place Y. wautersii and Y. pseudotuberculosis on the same branch, further substantiate this data. We propose the unification of Y. pseudotuberculosis and Y. wautersii as Y. pseudotuberculosis subsp. pseudotuberculosis and Y. pseudotuberculosis subsp. wautersii, respectively.

To harness the quantitative potential of next-generation sequencing for data normalization, spike-in controls are essential. We have engineered three bacterial genomes (Escherichia coli, Staphylococcus aureus, and Clostridium perfringens) to contain a unique synthetic DNA tag that can be detected via 16S rRNA profiling and whole genome sequencing assays. To demonstrate the utility of the spike-in control in microbiome studies, we mixed precise quantities of genomic DNA from the recombinant bacterial strains to create a genomic DNA spike-in standard. This quantified standard was spiked into a known mock community (ATCC® MSA-1000™) containing genomic DNA prepared from 10 different bacterial strains. The resulting data showed that the unique tag of all three bacteria was identifiable and quantifiable by shotgun and 16S rRNA amplicon sequencing using V1/V2, V3/V4, and V4 primers. Spiking these recombinant bacterial genomic DNA at an optimal concentration did not affect microbiome abundance. Further, we demonstrated that the spike-in standard was applicable as an internal control for absolute quantitation. These proof-of-concept experiments support the utility of using a spike-in control with a unique 16S rRNA tag to monitor the full process of a microbiome workflow for both 16S rRNA and shotgun metagenomics assays.

With over 2 million infections per year in the United States alone, antimicrobial resistance (AMR) among bacterial pathogens has become a serious threat to global health. To thwart the AMR threat via drug discovery and diagnostics research, accurate characterization of AMR gene clusters, mobile elements, insertions, and deletions in bacterial genomes is crucial. To that end, we developed the ATCC Global Priority Superbugs Collection, which comprises 57 fully authenticated, characterized, and sequenced strains representing critical level pathogens. Here, we discuss the phenotypic and genotypic characterization of these strains through antimicrobial susceptibility profiling and a standardized sequencing, assembly, and annotation pipeline.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with Congenital Zika Syndrome (CZS), which comprises a wide range of congenital abnormalities in fetuses and infants infected with ZIKV before birth (1 and 2). In a small number of patients, ZIKV infection is strongly associated with the neurological autoimmune disorder Gullian Barré Syndrome (GBS) (3). To date, no vaccines or antiviral strategies are licensed for Zika Virus. Our aim is the development of a novel Zika vaccine candidate safe from antibody-dependent enhancement (ADE).