third generation sequencing
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AbstractThe genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides an idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.

Background: While multiple genetic causes of movement disorders have been identified in the past decade, modifying factors of disease expression are still largely unknown for most conditions. X-linked dystonia-parkinsonism (XDP) is an inherited neurodegenerative disease caused by a SINE-VNTR-Alu (SVA)-type retrotransposon insertion that contains a hexanucleotide repeat within an intron of the TAF1 gene. To date, four putative genetic modifiers explain about 65% of variance in age at onset in XDP. However, additional genetic modifiers are conceivably at play in XDP and may include mismatches of the SVA hexanucleotide repeat motif. We aim to identify additional genetic modifiers of XDP expressivity and age at onset (AAO). Methods: Third-generation sequencing of PCR amplicons from XDP patients (n=202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment were used to confirm the presence of identified repeat mismatches. Results: An increased frequency of deletions at the beginning of the hexanucleotide repeat (CCCTCT)n domain was found. Specifically, three deletions at positions 11, 14, and 17 of the TAF1 SVA repeat motif of somatic mosaic origins were detected in different combinations. The most common one was three deletions (1-2-3) at a median frequency 0.425 (IQR:0.42-0.43) and deletions within positions 11 and 14 (1-2-wt) at a median frequency 0.128 (IQR:0.12-0.13). The frequency of deletions at positions 11 and 14 correlated with repeat number (r=-0.48, p=9.5x10-13) and AAO (r=0.34, p=9.5x10-7). The association with AAO still stands when including other modifier genotypes (MSH3 and PMS2) in a regression model. However, the association dissipates when including repeat numbers. Conclusion: We present a novel mosaic repeat motif deletion within the hexanucleotide repeat (CCCTCT)n domain of TAF1 SVA. Our study illustrates: 1) the importance of somatic mosaic genotypes; 2) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; 3) that these variations may remain undetected without assessment of single molecules.

Abstract Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

To analyze the whole genome of Bacillus aryabhattai strain SK1-7 and explore its potassium solubilization characteristics and mechanism, thus providing a theoretical basis for analyzing the utilization and improvement of insoluble potassium resources in soil. Genome information for Bacillus aryabhattai SK1-7 was obtained by using Illumina NovaSeq second-generation sequencing and GridION Nanopore ONT third-generation sequencing technology. The contents of organic acids and polysaccharides in fermentation broth of Bacillus aryabhattai SK1-7 were determined by high-performance liquid chromatography and the anthrone sulfuric acid method, and the expression levels of the potassium solubilization-related genes ackA, epsB, gltA, mdh and ppc were compared by real-time fluorescence quantitative PCR under different potassium source culture conditions. The whole genome of the strain consisted of a complete chromosome sequence and four plasmid sequences. The sequence sizes of the chromosomes and plasmids P1, P2, P3 and P4 were 5,188,391 bp, 136,204 bp, 124,862 bp, 67,200 bp and 12,374 bp, respectively. The GC contents were 38.2, 34.4, 33.6, 32.8, and 33.7%. Strain SK1-7 mainly secreted malic, formic, acetic and citric acids under culture with an insoluble potassium source. The polysaccharide content produced with an insoluble potassium source was higher than that with a soluble potassium source. The expression levels of five potassium solubilization-related genes with the insoluble potassium source were higher than those with the soluble potassium source.

Although next-generation sequencing (NGS) technology revolutionized sequencing, offering a tremendous sequencing capacity with groundbreaking depth and accuracy, it continues to demonstrate serious limitations. In the early 2010s, the introduction of a novel set of sequencing methodologies, presented by two platforms, Pacific Biosciences (PacBio) and Oxford Nanopore Sequencing (ONT), gave birth to third-generation sequencing (TGS). The innovative long-read technologies turn genome sequencing into an ease-of-handle procedure by greatly reducing the average time of library construction workflows and simplifying the process of de novo genome assembly due to the generation of long reads. Long sequencing reads produced by both TGS methodologies have already facilitated the decipherment of transcriptional profiling since they enable the identification of full-length transcripts without the need for assembly or the use of sophisticated bioinformatics tools. Long-read technologies have also provided new insights into the field of epitranscriptomics, by allowing the direct detection of RNA modifications on native RNA molecules. This review highlights the advantageous features of the newly introduced TGS technologies, discusses their limitations and provides an in-depth comparison regarding their scientific background and available protocols as well as their potential utility in research and clinical applications.

Metagenomics is a new approach to study microorganisms obtained from a specific environment by functional gene screening or sequencing analysis. Metagenomics studies focus on microbial diversity, community constitute, genetic and evolutionary relationships, functional activities, and interactions and relationships with the environment. Sequencing technologies have evolved from shotgun sequencing to high-throughput, next-generation sequencing (NGS), and third-generation sequencing (TGS). NGS and TGS have shown the advantage of rapid detection of pathogenic microorganisms. With the help of new algorithms, we can better perform the taxonomic profiling and gene prediction of microbial species. Functional metagenomics is helpful to screen new bioactive substances and new functional genes from microorganisms and microbial metabolites. In this article, basic steps, classification, and applications of metagenomics are reviewed.

Here we describe a powerful method, BIND&MODIFY, for probing histone modifications and transcription factors at single molecular level. Our approach used the recombinant fused protein A-M.EcoGII, which tethers the methyltransferase M.EcoGII to the protein binding sites and locally labels the neighboring DNA regions via artificial methylations. This method could reveal ingle-molecule heterogenous histone modification status and CpG methylation at the same time, and could enable quantify the correlation between the distal elements. Further applications based on this method's concept could be applied to probe multiple protein binding events on the same single molecular DNA. The method proposed herein may soon become an essential tool for third-generation sequencing in the future.

Abstract The polyketide griseofulvin is a natural antifungal compound and research in griseofulvin has been key in establishing our current understanding of polyketide biosynthesis. Nevertheless, the griseofulvin gsf biosynthetic gene cluster (BGC) remains poorly understood in most fungal species, including Penicillium griseofulvum where griseofulvin was first isolated. To elucidate essential genes involved in griseofulvin biosynthesis, we performed third-generation sequencing to obtain the genome of Penicillium griseofulvum strain D-756. Furthermore, we gathered publicly available genome of 11 other fungal species in which gsf gene cluster was identified. In a comparative genome analysis, we annotated and compared the gsf BGC of all 12 fungal genomes. Our findings show no gene rearrangements at the gsf BGC. Furthermore, seven gsf genes are conserved by most genomes surveyed whereas the remaining six were poorly conserved. This study provides new insights into differences between gsf BGC and suggests that seven gsf genes are essential in griseofulvin production.

Objectives: Infections of the ascitic fluid are serious conditions that require rapid diagnosis and treatment. Ascites is often accompanied by other critical pathologies such as gastrointestinal bleeding and bowel perforation, and infection increases the risk of mortality in intensive care patients. Owing to a relatively low success rate of conventional culture methods in identifying the responsible pathogens, new methods may be helpful to guide antimicrobial therapy and to refine empirical regimens. Here, we aim to assess outcomes and to identify responsible pathogens in ascitic fluid infections, in order to improve patients’ care and to guide empirical therapy. Methods: Between October 2019 and March 2021, we prospectively collected 50 ascitic fluid samples from ICU patients with suspected infection. Beside standard culture-based microbiology methods, excess fluid underwent DNA isolation and was analyzed by next- and third-generation sequencing (NGS) methods. Results: NGS-based methods had higher sensitivity in detecting additional pathogenic bacteria such as E. faecalis and Klebsiella in 33 out of 50 (66%) ascitic fluid samples compared with culture-based methods (26%). Anaerobic bacteria were especially identified by sequencing-based methods in 28 samples (56%), in comparison with only three samples in culture. Analysis of clinical data showed a correlation between sequencing results and various clinical parameters such as peritonitis and hospitalization outcomes. Conclusions: Our results show that, in ascitic fluid infections, NGS-based methods have a higher sensitivity for the identification of clinically relevant pathogens than standard microbiological culture diagnostics, especially in detecting hard-to-culture anaerobic bacteria. Patients with such infections may benefit from the use of NGS methods by the possibility of earlier and better targeted antimicrobial therapy, which has the potential to lower the high morbidity and mortality in critically ill patients with ascitic bacterial infection.

Abstract Background: Ticks are arthropods that can carry multiple pathogens and parasitize on livestock and mammals as well as on humans. Animal husbandry in Inner Mongolia, China, provides a suitable tick habitat. In this study, PacBio full-length 16S rDNA third-generation sequencing was used to analyze the diversity of microbial communities carried by ticks in different regions of Inner Mongolia. The aim of the study is to characterize the microbiome carried by ticks in different geographical locations and to provide theoretical support for regional prevention and control of pathogen populations in the future. Methods: In this study, a total of 905 Dermacentor nuttalli and 36 Ixodes persuleatus were collected from the surface of sheep in four main pasture areas in Inner Mongolia. Pooled DNA samples were prepared from three samples from each region and from each tick species. In total the microbial diversity of 12 samples was analyzed by PacBio full-length 16S rRNA third-generation sequencing, and the α and β diversity were determined. Results: The main bacterial genera we found were Rickettsia (35.27%), Ac37b (19.33%), Arsenophonus (11.21%), Candidatus Lariskella (10.84%), and Acinetobacter (7.17%). There were significant differences in the microbial composition of ticks from different regions and in different tick species. Rickettsia bellii was found in the I. persuleatus group. In addition, Anaplasma and a novel tyhpus group Rickettsia species (R. bacterium Ac37b) were found in the sample group of D. nuttalli in the city of Ordos.Conclusions: In this study, Rickettsia bellii was first found in I. persuleatus in Inner Mongolia, and a novel tyhpus group Rickettsia species (R. bacterium Ac37b) was found in D. nuttalli from the city of Ordos. Our study provides a basis for the prevention and control of tick-borne diseases through the analysis of tick microbial diversity in different regions of Inner Mongolia. Furthermore, we were able to detect a new tick-borne pathogen in D. nutalli.