Whole Genome Sequencing Approach Research Articles

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures.

Next-generation sequencing (NGS) yields powerful opportunities for studying human papillomavirus (HPV) genomics for applications in epidemiology, public health, and clinical diagnostics. HPV genotypes, variants, and point mutations can be investigated in clinical materials and described in previously unprecedented detail. However, both the NGS laboratory analysis and bioinformatical approach require numerous steps and checks to ensure robust interpretation of results. Here, we provide a step-by-step review of recommendations for validation and quality assurance procedures of each step in the typical NGS workflow, with a focus on whole-genome sequencing approaches. The use of directed pilots and protocols to ensure optimization of sequencing data yield, followed by curated bioinformatical procedures, is particularly emphasized. Finally, the storage and sharing of data sets are discussed. The development of international standards for quality assurance should be a goal for the HPV NGS community, similar to what has been developed for other areas of sequencing efforts including microbiology and molecular pathology. We thus propose that it is time for NGS to be included in the global efforts on quality assurance and improvement of HPV-based testing and diagnostics.

AmpliCoV: Rapid Whole-Genome Sequencing Using Multiplex PCR Amplification and Real-Time Oxford Nanopore MinION Sequencing Enables Rapid Variant Identification of SARS-CoV-2.

Since the emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the scientific community has been sharing data on epidemiology, diagnostic methods, and whole-genomic sequences almost in real time. The latter have already facilitated phylogenetic analyses, transmission chain tracking, protein modeling, the identification of possible therapeutic targets, timely risk assessment, and identification of novel variants. We have established and evaluated an amplification-based approach for whole-genome sequencing of SARS-CoV-2. It can be used on the miniature-sized and field-deployable sequencing device Oxford Nanopore MinION, with sequencing library preparation time of 10 min. We show that the generation of 50,000 total reads per sample is sufficient for a near complete coverage (>90%) of the SARS-CoV-2 genome directly from patient samples even if virus concentration is low (Ct 35, corresponding to approximately 5 genome copies per reaction). For patient samples with high viral load (Ct 18–24), generation of 50,000 reads in 1–2 h was shown to be sufficient for a genome coverage of >90%. Comparison to Illumina data reveals an accuracy that suffices to identify virus mutants. AmpliCoV can be applied whenever sequence information on SARS-CoV-2 is required rapidly, for instance for the identification of circulating virus mutants.

Copy number alteration profiling facilitates differential diagnosis between ossifying fibroma and fibrous dysplasia of the jaws

Ossifying fibroma (OF) and fibrous dysplasia (FD) are two fibro-osseous lesions with overlapping clinicopathological features, making diagnosis challenging. In this study, we applied a whole-genome shallow sequencing approach to facilitate differential diagnosis via precise profiling of copy number alterations (CNAs) using minute amounts of DNA extracted from morphologically correlated microdissected tissue samples. Freshly frozen tissue specimens from OF (n = 29) and FD (n = 28) patients were obtained for analysis. Lesion fibrous tissues and surrounding normal tissues were obtained by laser capture microdissection (LCM), with ~30–50 cells (5 000–10 000 µm2) per sample. We found that the rate of recurrent CNAs in OF cases was much higher (44.8%, 13 of 29) than that in FD cases (3.6%, 1 of 28). Sixty-nine percent (9 of 13) of the CNA-containing OF cases involved segmental amplifications and deletions on Chrs 7 and 12. We also identified eight CNA-associated genes (HILPDA, CALD1, C1GALT1, MICALL2, PHF14, AIMP2, MDM2, and CDK4) with amplified expression, which was consistent with the copy number changes. We further confirmed a jaw lesion with a previous uncertain diagnosis due to its ambiguous morphological features and the absence of GNAS mutation as OF based on the typical Chr 12 amplification pattern in its CNA profile. Moreover, analysis of a set of longitudinal samples collected from an individual with a cellular lesion in suspicion of OF at the first surgery, recurrence and the latest malignant transformation revealed identical CNA patterns at the three time points, suggesting that copy number profiling can be used as an important tool to identify borderline lesions or lesions with malignant potential. Overall, CNA profiling of fibro-osseous lesions can greatly improve differential diagnosis between OF and FD and help predict disease progression.

MICROEVOLUTION OF DESULFOVIBRIO VULGARIS CO-CULTURED WITH METHANOSARCINA BARKERI REVEALED BY GENOME RE-SEQUENCING AND SINGLE-CELL RT-QPCR ANALYSIS

An integrative approach of adaptive laboratory evolution, whole-genome sequencing and single-cell analysis was used to explore mechanisms related to establishment and maintenance of syntrophic interaction between sulfate-reducing Desulfovibrio vulgaris and methanogen Methanosarcina barkeri. Adaptive laboratory evolution of the D. vulgaris and M. barkeri dual-cultures under two different concentrations of electron donor lactate (38 mM and 50 mM) was conducted by propagating continuously for 50 transfers (~200 generations). Physiological analysis showed that, compared with the initial dual-cultures, the adapted dual-cultures (E38 and E50) have increased growth rates (1.1-fold and 1.2 -fold) and higher biomass yields (3.0-fold and 3.8-fold) on 38 mM and 50 mM lactate, respectively. Whole-genome re-sequencing of D. vulgaris in the adapted dual-cultures revealed 11 and 12 mutations in the D. vulgaris genomes of E38 and E50 dual-cultures, respectively, among which 4 mutations were found in both adapted dual-cultures. RT-qPCR analysis showed that the expression levels of 8 mutated genes were gradually up-regulated in D. vulgaris along with the evolution process. In addition, their heterogeneity was found decreased along with the evolution, as revealed by single-cell RT-qPCR analysis, reflecting adjustments of both gene expression and gene heterogeneity to the gradually established syntrophic relationship.

Integration of population and functional genomics to understand mechanisms of artemisinin resistance in Plasmodium falciparum

Resistance to antimalarial drugs, and in particular to the artemisinin derivatives and their partner drugs, threatens recent progress toward regional malaria elimination and eventual global malaria eradication. Population-level studies utilizing whole-genome sequencing approaches have facilitated the identification of regions of the parasite genome associated with both clinical and in vitro drug-resistance phenotypes. However, the biological relevance of genes identified in these analyses and the establishment of a causal relationship between genotype and phenotype requires functional characterization. Here we examined data from population genomic and transcriptomic studies in the context of data generated from recent functional studies, using a new population genetic approach designed to identify potential favored mutations within the region of a selective sweep (iSAFE). We identified several genes functioning in pathways now known to be associated with artemisinin resistance that were supported in early population genomic studies, as well as potential new drug targets/pathways for further validation and consideration for treatment of artemisinin-resistant Plasmodium falciparum. In addition, we establish the utility of iSAFE in identifying positively-selected mutations in population genomic studies, potentially accelerating the time to functional validation of candidate genes.

Lactobacillus casei CCFM1074 Alleviates Collagen-Induced Arthritis in Rats via Balancing Treg/Th17 and Modulating the Metabolites and Gut Microbiota

Gut microbiota and their influence on metabolites are receiving increasing attentions in autoimmune diseases including rheumatoid arthritis (RA). Probiotics become a promising manipulator to prevent or attenuate the progression of arthritis, some evidences suggesting that lactobacilli treatment influence the responses to RA therapy but the underlying mechanisms are limited. By using a collagen-induced arthritis (CIA) rats, the study assessed the effects of two L. casei strains (CCFM1074, CCFM1075) on the immune responses, gut microbiota and plasma metabolites via an integrated cross-omics approach including fecal 16S rRNA high-throughput sequencing and plasma metabolomics. The genome of the two strains was analyzed and compared using whole-genome sequencing approach to further confirm biology functions. CCFM1074 reduced arthritic symptoms while CCFM1075 did not, though both strains down-regulated the plasma IL-6 and Th17 cells proportion. CCFM1074 enhanced the proportion of Treg cells in mesenteric lymph nodes which was significantly associated with SCFAs upregulation, as well as with genomic evidence that CCFM1074 possesses more functional genes involved in carbohydrate metabolism. Moreover, CCFM1074 regulated the gut microbiota, including modulating community structure, decreasing the abundance of Alistipes and Parabacteroides and increasing the abundance of Oscillibacter. The differential metabolites modulated by CCFM1074 including eicosapentaenoic acid and docosapentaenoic acid which involved in unsaturated fatty acids metabolism. Furthermore, alterations of gut microbial community were correlated with the plasma metabolome. In summary, L. casei CCFM1074 alleviated arthritis via rebalancing gut microbiota, immune responses and plasma metabolites.

Comparison of structural variants detected by optical mapping with long-read next-generation sequencing.

Recent studies have shown the potential of using long-read whole-genome sequencing (WGS) approaches and optical mapping (OM) for the detection of clinically relevant structural variants (SVs) in cancer research. Three main long-read WGS platforms are currently in use: Pacific Biosciences (PacBio), Oxford Nanopore Technologies (ONT) and 10x Genomics. Recently, whole-genome OM technology (Bionano Genomics) has been introduced into human diagnostics. Questions remain about the accuracy of these long-read sequencing platforms, how comparable/interchangeable they are when searching for SVs and to what extent they can be replaced or supplemented by OM. Moreover, no tool can effectively compare SVs obtained by OM and WGS. This study compared optical maps of the breast cancer cell line SKBR3 with AnnotSV outputs from WGS platforms. For this purpose, a software tool with comparative and filtering features was developed. The majority of SVs up to a 50 kbp distance variance threshold found by OM were confirmed by all WGS platforms, and ∼99% of translocations and ∼80% of deletions found by OM were confirmed by both PacBio and ONT, with ∼70% being confirmed by 10x Genomics in combination with PacBio and/or ONT. Interestingly, long deletions (>100 kbp) were detected only by 10x Genomics. Regarding insertions, ∼74% was confirmed by PacBio and ONT, but none by 10x Genomics. Inversions and duplications detected by OM were not detected by WGS. Moreover, the tool enabled the confirmation of SVs that overlapped in the same gene(s) and was applied to the filtering of disease-associated SVs. https://github.com/novosadt/om-annotsv-svc.

Global analysis of transcription remodeling in heart and brain in a rat model of heart failure‐induced by myocardial infarction

Myocardial infarction (MI)-induced heart failure (HF) is a devastating disease with high incidences that threats millions of patients in the U.S. MI-induced HF causes irreversible injury to the heart as well as many other organs affected by insufficient blood supply. The impaired cardiac function in HF also evokes neural and humoral compensatory mechanisms to maintain blood pressure and the performance of heart. Permanent damages in these organs and subsequent compensatory responses caused by MI may result in transcriptional modification or epigenetic changes in chromatins of relevant regions in the development of HF. However, the alternations of gene expression affected by MI in HF is not completely understood. The present study sought to determine the transcription landscape remodeling and adaptive epigenetic alternation in MI-induced HF rat. To gain insights into global transcriptional changes, we used an unbiased whole-genome RNA sequencing approach to interrogate the transcriptional differences in heart and brain in MI-induced HF vs. sham rats. Male Sprague-Dawley rats underwent left anterior descending (LAD) coronary artery ligation to induce HF or sham operation (Sham). Myocardial infarction in HF was confirmed by echocardiography. Heart and brain were collected two weeks after LAD ligation. The total RNA was extracted from myocardial tissues adjacent to the infracted zone and hypothalamic paraventricular nucleus (PVN), an important cardiovascular/autonomic brain. Principle component analysis (PCA) shows that the heart and PVN samples of HF rats (n=4) were well separated from their counterparts of Sham rats (n=4), respectively. We detected 865 upregulated and 245 downregulated differentially expressed genes (DEGs) in the heart (p value cut-off is set to <0.01, and threshold of fold change cut-off is set to >2 for upregulation or <1/2 for downregulation), while 35 upregulated and 39 downregulated DEGs were identified in PVN in HF animals (p value cut-off is set to <0.05, and threshold of fold change cut-off is set to >1.2 for upregulation or <0.8 for downregulation). Moreover, enrichment analysis using gene ontology database KEGG (Kyoto Encyclopedia of Genes and Genomes) has identified 25 pathways in the heart. Each pathway contains 9-34 DEGs. Meanwhile, MI-induced DEGs in PVN are categorized into several pathways that are associated with angiotensin and angiogenesis (e.g. aplnr), MAP kinase signaling cascades (e.g. map3k6), hypoxia responses (e.g. Hif3a), neural activity and inflammation/immune activation, et. al. Further study will determine the functional significance of these DEGs in HF. Collectively, these data reveal a regulatory feedback loop along the heart-brain axis and suggest a previously unrecognized transcriptional remodeling mechanism in the brain that may contribute to the neurohormonal and autonomic regulation in HF.

Genome-Wide Identification of Sequence Variations and SSR Marker Development in the Munake Grape Cultivar

The Munake grape cultivar produces uniquely flavored and high-quality fruits. Despite the numerous beneficial agronomic traits of Munake, there are few genetic resources available for this cultivar. To address this knowledge gap, the entire genome was sequenced using whole-genome sequencing approaches and compared with a Vitis vinifera L. reference genome. This study describes more than 3 million single nucleotide polymorphism (SNP), 300,000 insertion and deletion (InDel), 14,000 structural variation (SV), and 80,000 simple sequence repeat (SSR) markers (one SSR per 4.23 kb), as well as their primers. Among the SSRs, 44 SSR primer pairs were randomly selected and validated by polymerase chain reaction (PCR), allowing discrimination between the different Munake cultivar genotypes. The genetic data provided allow a deeper understanding of Munake cultivar genomic sequence and contribute to better knowledge of the genetic basis behind its key agronomic traits.

An Amplicon-Based Approach for the Whole-Genome Sequencing of Human Metapneumovirus.

Human metapneumovirus (HMPV) is an important cause of upper and lower respiratory tract disease in individuals of all ages. It is estimated that most individuals will be infected by HMPV by the age of five years old. Despite this burden of disease, there remain caveats in our knowledge of global genetic diversity due to a lack of HMPV sequencing, particularly at the whole-genome scale. The purpose of this study was to create a simple and robust approach for HMPV whole-genome sequencing to be used for genomic epidemiological studies. To design our assay, all available HMPV full-length genome sequences were downloaded from the National Center for Biotechnology Information (NCBI) GenBank database and used to design four primer sets to amplify long, overlapping amplicons spanning the viral genome and, importantly, specific to all known HMPV subtypes. These amplicons were then pooled and sequenced on an Illumina iSeq 100 (Illumina, San Diego, CA, USA); however, the approach is suitable to other common sequencing platforms. We demonstrate the utility of this method using a representative subset of clinical samples and examine these sequences using a phylogenetic approach. Here we present an amplicon-based method for the whole-genome sequencing of HMPV from clinical extracts that can be used to better inform genomic studies of HMPV epidemiology and evolution.

Genome Sequencing as an Alternative to Cytogenetic Analysis in Myeloid Cancers

BackgroundGenomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated.MethodsWe used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing.ResultsWhole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different.ConclusionsIn our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.)

Exploring the Composition and Functions of Plastic Microbiome Using Whole-Genome Sequencing.

Besides the ecotoxicological consequences of microplastics and associated chemicals, the association of microbes on plastics has greater environmental implications as microplastics may select for unique microbiome participating in environmentally significant functions. Despite this, the functional potential of the microbiome associated with different types of plastics is understudied. Here, we investigate the interaction between plastic and marine biofilm-forming microorganisms through a whole-genome sequencing approach on four types of microplastics incubated in the marine environment. Taxonomic analysis suggested that the microplastic surfaces exhibit unique microbial profiles and niche partitioning among the substrates. In particular, the abundance of Vibrio alginolyticus and Vibrio campbellii suggested that microplastic pollution may pose a potential risk to the marine food chain and negatively impact aquaculture industries. Microbial genera involved in xenobiotic compound degradation, carbon cycling, and genes associated with the type IV secretion system, conjugal transfer protein TraG, plant-pathogen interaction, CusA/CzcA family heavy metal efflux transfer proteins, and TolC family proteins were significantly enriched on all the substrates, indicating the variety of processes operated by the plastic-microbiome. The present study gives a detailed characterization of the rapidly altering microbial composition and gene pools on plastics and adds new knowledge surrounding the environmental ramifications of marine plastic pollution.

In Vitro Reassortment between Endemic Bluetongue Viruses Features Global Shifts in Segment Frequencies and Preferred Segment Combinations.

Bluetongue virus (BTV) is an arthropod-borne pathogen that is associated with sometimes severe disease in both domestic and wild ruminants. Predominantly transmitted by Culicoides spp. biting midges, BTV is composed of a segmented, double-stranded RNA genome. Vector expansion and viral genetic changes, such as reassortment between BTV strains, have been implicated as potential drivers of ongoing BTV expansion into previously BTV-free regions. We used an in vitro system to investigate the extent and flexibility of reassortment that can occur between two BTV strains that are considered enzootic to the USA, BTV-2 and BTV-10. Whole genome sequencing (WGS) was coupled with plaque isolation and a novel, amplicon-based sequencing approach to quantitate the viral genetic diversity generated across multiple generations of in vitro propagation. We found that BTV-2 and BTV-10 were able to reassort across multiple segments, but that a preferred BTV-2 viral backbone emerged in later passages and that certain segments were more likely to be found in reassortant progeny. Our findings indicate that there may be preferred segment combinations that emerge during BTV reassortment. Moreover, our work demonstrates the usefulness of WGS and amplicon-based sequencing approaches to improve understanding of the dynamics of reassortment among segmented viruses such as BTV.

Mapping of susceptible variants for cold medicine-related Stevens\u2013Johnson syndrome by whole-genome resequencing

Stevens–Johnson syndrome (SJS) and its severe condition with extensive skin detachment and a poor prognosis, toxic epidermal necrolysis (TEN), are immunologically mediated severe cutaneous reactions of the skin and mucous membranes such as the ocular surface. Genetic variations on the HLA-A and other autosomal genes have been identified as risk factors for cold medicine-related SJS/TEN with severe ocular complications (CM-SJS/TEN with SOC). Using a whole-genome sequencing (WGS) approach, we explored other susceptible variants of CM-SJS/TEN with SOC, especially among rare variants and structural variants (SVs). WGS was performed on samples from 133 patients with CM-SJS/TEN with SOC and 418 healthy controls to obtain single nucleotide polymorphisms (SNPs) and SVs. Genome-wide association tests were performed with these variants. Our genome-wide association test reproduced the associations of the common variants of HLA-A and loci on chromosome 16q12.1. We also identified novel associations of SVs on these loci and an aggregation of rare coding variants on the TPRM8 gene. In silico gene expression analysis on the HLA-A locus revealed that the SNP (rs12202296), which was significantly associated with susceptibility to CM-SJS/TEN with SOC, was correlated to an increase in HLA-A expression levels in the whole blood (P = 2.9 × 10−17), from the GTEx database. The majority of variants that were significantly associated with CM-SJS/TEN with SOC were found in non-coding regions, indicating the regulatory role of genetic variations in the pathogenesis of CM-SJS/TEN with SOC.

153. pilot Study of a Novel Whole-genome Sequencing Based Rapid Bacterial Identification Assay in Patients with Bacteremia

BackgroundBlood stream infections (BSI) are among the leading cause of morbidity and mortality, yet gold standard culture-based diagnostics have limited ability to guide therapeutic intervention due to multi-day turnaround time and low sensitivity. Day Zero Diagnostics has developed Blood2Bac™, a culture-free, species agnostic process to enrich bacteria direct from whole blood. Coupled with whole genome sequencing (WGS) and Day Zero Diagnostics’ Keynome® algorithmic tools for species ID and antimicrobial resistance (AMR), we conducted the first proof-of-concept feasibility study in an inpatient clinical setting.MethodsStudy participants were enrolled and specimens collected from Boston Medical Center. Eligibility criteria included hospitalized adults with suspected and/or documented BSI, irrespective of empiric antibiotic therapy duration. Whole blood samples were processed with Blood2Bac, sequenced on a nanopore platform, and bacterial ID determined with Keynome ID. Keynome ID results were compared with blood culture results to measure concordance.ResultsSpecimens from 21 participants were processed with Blood2Bac and nanopore sequencing. For 20/21 samples, Keynome ID calls were concordant with clinical blood culture, where 6 concordant positive and 14 were concordant negative. In 3 concordant samples, Keynome ID called positive while concurrent blood cultures were negative. However, all IDs corresponded with positive blood culture results from the day prior, suggesting potentially higher sensitivity for the Blood2Bac compared to blood culture. Two concordant positive IDs, resulted in >95% of the genome recovered and Keynome concomitantly resulted in AMR predictions with 100% accuracy compared to pathogen phenotype. In 1 discordant specimen, the Keynome ID result was negative while blood cultures 8 hours before were positive. In this case, the patient was on empiric therapy for 8 days prior to samples collection and cultures were negative 19-hours post specimen collection.ConclusionThese results highlight the sensitivity of a real-time blood WGS approach to identify BSI and its utility as a diagnostic to minimize unnecessary antibiotic exposure contributing to the antibiotic resistance crisis.Disclosures Archana Asundi, MD, Gilead (Scientific Research Study Investigator)Merck (Scientific Research Study Investigator)ViiV (Scientific Research Study Investigator) Nicole Billings, PhD, Day Zero Diagnostics (Employee) Zoe H. Rogers, MPH, Day Zero Diagnostics (Employee, Shareholder) Lisa S. Cunden, PhD, Day Zero Diagnostics (Shareholder) Imaly A. Nanayakkara, PhD, Day Zero Diagnostics (Employee, Shareholder) Chiahao Tsui, n/a, Day Zero Diagnostics (Employee, Shareholder) Paul Knysh, PhD, Day Zero Diagnostics (Employee) Cabell Maddux, n/a, Day Zero Diagnostics (Employee, Shareholder) Zachary Munro, n/a, Day Zero Diagnostics Inc. (Employee, Shareholder) Ian Herriott, BS, Day Zero Diagnostics (Employee, Shareholder) Miriam Huntley, PhD, Day Zero Diagnostics (Employee, Shareholder) Nina H. Lin, MD, Gilead Sciences (Scientific Research Study Investigator)ViiV (Scientific Research Study Investigator)

Molecular epidemiology in the HIV and SARS-CoV-2 pandemics.

Purpose of reviewThe aim of this review was to compare and contrast the application of molecular epidemiology approaches for the improved management and understanding of the HIV versus SARS-CoV-2 epidemics.Recent findingsMolecular biology approaches, including PCR and whole genome sequencing (WGS), have become powerful tools for epidemiological investigation. PCR approaches form the basis for many high-sensitivity diagnostic tests and can supplement traditional contact tracing and surveillance strategies to define risk networks and transmission patterns. WGS approaches can further define the causative agents of disease, trace the origins of the pathogen, and clarify routes of transmission. When coupled with clinical datasets, such as electronic medical record data, these approaches can investigate co-correlates of disease and pathogenesis. In the ongoing HIV epidemic, these approaches have been effectively deployed to identify treatment gaps, transmission clusters and risk factors, though significant barriers to rapid or real-time implementation remain critical to overcome. Likewise, these approaches have been successful in addressing some questions of SARS-CoV-2 transmission and pathogenesis, but the nature and rapid spread of the virus have posed additional challenges.SummaryOverall, molecular epidemiology approaches offer unique advantages and challenges that complement traditional epidemiological tools for the improved understanding and management of epidemics.

High quality genome assemblies of Mycoplasma bovis using a taxon-specific Bonito basecaller for MinION and Flongle long-read nanopore sequencing

BackgroundImplementation of Third-Generation Sequencing approaches for Whole Genome Sequencing (WGS) all-in-one diagnostics in human and veterinary medicine, requires the rapid and accurate generation of consensus genomes. Over the last years, Oxford Nanopore Technologies (ONT) released various new devices (e.g. the Flongle R9.4.1 flow cell) and bioinformatics tools (e.g. the in 2019-released Bonito basecaller), allowing cheap and user-friendly cost-efficient introduction in various NGS workflows. While single read, overall consensus accuracies, and completeness of genome sequences has been improved dramatically, further improvements are required when working with non-frequently sequenced organisms like Mycoplasma bovis. As an important primary respiratory pathogen in cattle, rapid M. bovis diagnostics is crucial to allow timely and targeted disease control and prevention. Current complete diagnostics (including identification, strain typing, and antimicrobial resistance (AMR) detection) require combined culture-based and molecular approaches, of which the first can take 1–2 weeks. At present, cheap and quick long read all-in-one WGS approaches can only be implemented if increased accuracies and genome completeness can be obtained.ResultsHere, a taxon-specific custom-trained Bonito v.0.1.3 basecalling model (custom-pg45) was implemented in various WGS assembly bioinformatics pipelines. Using MinION sequencing data, we showed improved consensus accuracies up to Q45.2 and Q46.7 for reference-based and Canu de novo assembled M. bovis genomes, respectively. Furthermore, the custom-pg45 model resulted in mean consensus accuracies of Q45.0 and genome completeness of 94.6% for nine M. bovis field strains. Improvements were also observed for the single-use Flongle sequencer (mean Q36.0 accuracies and 80.3% genome completeness).ConclusionsThese results implicate that taxon-specific basecalling of MinION and single-use Flongle Nanopore long reads are of great value to be implemented in rapid all-in-one WGS tools as evidenced for Mycoplasma bovis as an example.

Genome sequence of an uncharted halophilic bacterium Robertkochia marina with deciphering its phosphate-solubilizing ability.

The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30DT is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land.

Extreme enrichment of VNTR-associated polymorphicity in human subtelomeres: genes with most VNTRs are predominantly expressed in the brain

The human genome harbors numerous structural variants (SVs) which, due to their repetitive nature, are currently underexplored in short-read whole-genome sequencing approaches. Using single-molecule, real-time (SMRT) long-read sequencing technology in combination with FALCON-Unzip, we generated a de novo assembly of the diploid genome of a 115-year-old Dutch cognitively healthy woman. We combined this assembly with two previously published haploid assemblies (CHM1 and CHM13) and the GRCh38 reference genome to create a compendium of SVs that occur across five independent human haplotypes using the graph-based multi-genome aligner REVEAL. Across these five haplotypes, we detected 31,680 euchromatic SVs (>50 bp). Of these, ~62% were comprised of repetitive sequences with ‘variable number tandem repeats’ (VNTRs), ~10% were mobile elements (Alu, L1, and SVA), while the remaining variants were inversions and indels. We observed that VNTRs with GC-content >60% and repeat patterns longer than 15 bp were 21-fold enriched in the subtelomeric regions (within 5 Mb of the ends of chromosome arms). VNTR lengths can expand to exceed a critical length which is associated with impaired gene transcription. The genes that contained most VNTRs, of which PTPRN2 and DLGAP2 are the most prominent examples, were found to be predominantly expressed in the brain and associated with a wide variety of neurological disorders. Repeat-induced variation represents a sizeable fraction of the genetic variation in human genomes and should be included in investigations of genetic factors associated with phenotypic traits, specifically those associated with neurological disorders. We make available the long and short-read sequence data of the supercentenarian genome, and a compendium of SVs as identified across 5 human haplotypes.

Evaluation of an angiotensin Type 1 receptor blocker on the reconsolidation of fear memory

Inhibition of the angiotensin type 1 receptor (AT1R) has been shown to decrease fear responses in both humans and rodents. These effects are attributed to modulation of extinction learning, however the contribution of AT1R to alternative memory processes remains unclear. Using classic Pavlovian conditioning combined with radiotelemetry and whole-genome RNA sequencing, we evaluated the effects of the AT1R antagonist losartan on fear memory reconsolidation. Following the retrieval of conditioned auditory fear memory, animals were given a single intraperitoneal injection of losartan or saline. In response to the conditioned stimulus (CS), losartan-treated animals exhibited significantly less freezing at 24 h and 1 week; an effect that was dependent upon memory reactivation and independent of conditioned cardiovascular reactivity. Using an unbiased whole-genome RNA sequencing approach, transcriptomic analysis of the basolateral amygdala (BLA) identified losartan-dependent differences in gene expression during the reconsolidation phase. These findings demonstrate that post-retrieval losartan modifies behavioral and transcriptomic markers of conditioned fear memory, supporting an important regulatory role for this receptor in reconsolidation and as a potential pharmacotherapeutic target for maladaptive fear disorders such as PTSD.