A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.
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ABSTRACT:
SUBMITTER: Reteng P
PROVIDER: S-EPMC8463598 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We report a case of murine typhus in China caused by Rickettsia typhi and diagnosed by nanopore targeted sequencing of a bronchoalveolar lavage fluid sample. This case highlights that nanopore targeted sequencing can effectively detect clinically unexplained infections and be especially useful for detecting infections in patients without typical signs and symptoms.
| S-EPMC10202882 | biostudies-literature
Project description:ObjectivesTo develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs).MethodsReal-time PCR (qPCR) was initially performed to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections among a total of 200 vulvo-vaginal swab samples from female sex workers in Ecuador. qPCR positive samples plus qPCR negative controls for these STIs were subjected to single gene targeted PCR MinION-nanopore sequencing using the smartphone operated MinIT.ResultsAmong 200 vulvo-vaginal swab samples 43 were qPCR positive for at least one of the STIs. Single gene targeted nanopore sequencing generally yielded higher pathogen specific read counts in qPCR positive samples than qPCR negative controls. Of the 26 CT, NG or MG infections identified by qPCR, 25 were clearly distinguishable from qPCR negative controls by read count. Discrimination of TV qPCR positives from qPCR negative controls was poorer as many had low pathogen loads (qPCR cycle threshold >35) which produced few specific reads. Real-time AMR profiling revealed that 3/3 NG samples identified had gyrA mutations associated with fluoroquinolone resistance, 2/10 of TV had mutations related to metronidazole resistance, while none of the MG samples possessed 23S rRNA gene mutations contributing to macrolide resistance.ConclusionsSingle gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common genital STIs shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.
| S-EPMC8782522 | biostudies-literature
Project description:Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.
| S-EPMC6947272 | biostudies-literature
Project description:BackgroundFlaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods.MethodsWe developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates.ResultsThe novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates.ConclusionThis PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.
| S-EPMC8442469 | biostudies-literature
Project description:BackgroundResistance to chemotherapy is the most common cause of treatment failure in acute myeloid leukemia (AML) and the drug efflux pump ABCB1 is a critical mediator. Recent studies have identified promoter translocations as common drivers of high ABCB1 expression in recurrent, chemotherapy-treated high-grade serous ovarian cancer (HGSC) and breast cancer. These fusions place ABCB1 under the control of a strong promoter while leaving its open reading frame intact. The mechanisms controlling high ABCB1 expression in AML are largely unknown. We therefore established an experimental system and analysis pipeline to determine whether promoter translocations account for high ABCB1 expression in cases of relapsed human AML.MethodsThe human AML cell line THP-1 was used to create a model of chemotherapy resistance in which ABCB1 expression was driven by a promoter fusion. The THP-1 model was used to establish a targeted nanopore long-read sequencing approach that was then applied to cases of ABCB1high HGSC and AML. H3K27Ac ChIP sequencing was used to assess the activity of native promoters in cases of ABCB1high AML.ResultsProlonged in vitro daunorubicin exposure induced activating ABCB1 promoter translocations in human THP-1 AML cells, similar to those recently described in recurrent high-grade serous ovarian and breast cancers. Targeted nanopore sequencing proved an efficient method for identifying ABCB1 structural variants in THP-1 AML cells and HGSC; the promoter translocations identified in HGSC were both previously described and novel. In contrast, activating ABCB1 promoter translocations were not identified in ABCB1high AML; instead H3K27Ac ChIP sequencing demonstrated active native promoters in all cases studied.ConclusionsDespite frequent high level expression of ABCB1 in relapsed primary AML we found no evidence of ABCB1 translocations and instead confirmed high-level activity of native ABCB1 promoters, consistent with endogenous regulation.
| S-EPMC7654162 | biostudies-literature
Project description:Among molecular-based techniques for fungal identification, Sanger sequencing of the primary universal fungal DNA barcode, the internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), is commonly used in clinical routine laboratories due to its simplicity, universality, efficacy, and affordability for fungal species identification. However, Sanger sequencing fails to identify mixed ITS sequences in the case of mixed infections. To overcome this limitation, different high-throughput sequencing technologies have been explored. The nanopore-based technology is now one of the most promising long-read sequencing technologies on the market as it has the potential to sequence the full-length ITS region in a single read. In this study, we established a workflow for species identification using the sequences of the entire ITS region generated by nanopore sequencing of both pure yeast isolates and mocked mixed species reads generated with different scenarios. The species used in this study included Candida albicans (n = 2), Candida tropicalis (n = 1), Nakaseomyces glabratus (formerly Candida glabrata) (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (formerly Candida krusei) (n = 1), and Cryptococcus neoformans (n = 1). Comparing various methods to generate the consensus sequence for fungal species identification, the results from this study indicate that read clustering using a modified version of the NanoCLUST pipeline is more sensitive than Canu or VSEARCH, as it classified species accurately with a lower abundance cluster of reads (3% abundance compared to 10% with VSEARCH). The modified NanoCLUST also reduced the number of classified clusters compared to VSEARCH, making the subsequent BLAST+ analysis faster. Subsampling of the datasets, which reduces the size of the datasets by approximately tenfold, did not significantly affect the identification results in terms of the identified species name, percent identity, query coverage, percentage of reads in the classified cluster, and the number of clusters. The ability of the method to distinguish mixed species within sub-populations of large datasets has the potential to aid computer analysis by reducing the required processing power. The herein presented new sequence analysis pipeline will facilitate better interpretation of fungal sequence data for species identification.
| S-EPMC10483712 | biostudies-literature
Project description:BackgroundBlood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources.MethodsWe used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method.ResultsComplete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively.ConclusionsOur findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel 'phylogenetic capture' methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens.
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Project description:SNPs, abundant in human genome with lower mutation rate, are attractive to genetic application like forensic, anthropological and evolutionary studies. Universal SNPs showing little allelic frequency variation among populations while remaining highly informative for human identification were obtained from previous studies. However, genotyping tools target only dozens of markers simultaneously, limiting their applications. Here, 124 SNPs were simultaneous tested using Ampliseq technology with Ion Torrent PGM platform. Concordance study was performed with 2 reference samples of 9947A and 9948 between NGS and Sanger sequencing. Full concordance were obtained except genotype of rs576261 with 9947A. Parameter of FMAR (%) was introduced for NGS data analysis for the first time, evaluating allelic performance, sensitivity testing and mixture testing. FMAR values for accurate heterozygotes should be range from 50% to 60%, for homozygotes or Y-SNP should be above 90%. SNPs of rs7520386, rs4530059, rs214955, rs1523537, rs2342747, rs576261 and rs12997453 were recognized as poorly performing loci, either with allelic imbalance or with lower coverage. Sensitivity testing demonstrated that with DNA range from 10 ng-0.5 ng, all correct genotypes were obtained. For mixture testing, a clear linear correlation (R(2) = 0.9429) between the excepted FMAR and observed FMAR values of mixtures was observed.
| S-EPMC4687036 | biostudies-literature
Project description:Ribosomal RNAs are decorated by numerous post-transcriptional modifications whose exact roles in ribosome biogenesis, function, and human pathophysiology remain largely unknown. Here, we report a targeted direct rRNA sequencing approach involving a substrate selection step and demonstrate its suitability to identify differential modification sites in combination with the JACUSA2 software. We compared JACUSA2 to other tools designed for RNA modification detection and show that JACUSA2 outperforms other software with regard to detection of base modifications such as methylation, acetylation and aminocarboxypropylation. To illustrate its widespread usability, we applied our method to a collection of CRISPR-Cas9 engineered colon carcinoma cells lacking specific enzymatic activities responsible for particular rRNA modifications and systematically compared them to isogenic wild-type RNAs. Besides the numerous 2'-O methylated riboses and pseudouridylated residues, our approach was suitable to reliably identify differential base methylation and acetylation events. Importantly, our method does not require any prior knowledge of modification sites or the need to train complex models. We further report for the first time detection of human rRNA modifications by direct RNA-sequencing on Flongle flow cells, the smallest-scale nanopore flow cell available to date. The use of these smaller flow cells reduces RNA input requirements, making our workflow suitable for the analysis of samples with limited availability and clinical work.
| S-EPMC10464549 | biostudies-literature
Project description:Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1-4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3??g of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.
| S-EPMC7145730 | biostudies-literature