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A novel quantitative targeted analysis of X-chromosome inactivation (XCI) using nanopore sequencing.

ABSTRACT: X-chromosome inactivation (XCI) analyses often assist in diagnostics of X-linked traits, however accurate assessment remains challenging with current methods. We developed a novel strategy using amplification-free Cas9 enrichment and Oxford nanopore technologies sequencing called XCI-ONT, to investigate and rigorously quantify XCI in human androgen receptor gene (AR) and human X-linked retinitis pigmentosa 2 gene (RP2). XCI-ONT measures methylation over 116 CpGs in AR and 58 CpGs in RP2, and separate parental X-chromosomes without PCR bias. We show the usefulness of the XCI-ONT strategy over the PCR-based golden standard XCI technique that only investigates one or two CpGs per gene. The results highlight the limitations of using the golden standard technique when the XCI pattern is partially skewed and the advantages of XCI-ONT to rigorously quantify XCI. This study provides a universal XCI-method on DNA, which is highly valuable in clinical and research framework of X-linked traits.

SUBMITTER: Johansson J

PROVIDER: S-EPMC10409790 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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A novel quantitative targeted analysis of X-chromosome inactivation (XCI) using nanopore sequencing.

Johansson Josefin J Lidéus Sarah S Höijer Ida I Ameur Adam A Gudmundsson Sanna S Annerén Göran G Bondeson Marie-Louise ML Wilbe Maria M

Scientific reports 20230808 1

X-chromosome inactivation (XCI) analyses often assist in diagnostics of X-linked traits, however accurate assessment remains challenging with current methods. We developed a novel strategy using amplification-free Cas9 enrichment and Oxford nanopore technologies sequencing called XCI-ONT, to investigate and rigorously quantify XCI in human androgen receptor gene (AR) and human X-linked retinitis pigmentosa 2 gene (RP2). XCI-ONT measures methylation over 116 CpGs in AR and 58 CpGs in RP2, and sep ...[more]

PMID: 37553382

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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.

Project description:BackgroundInfective endocarditis (IE) can be caused by a variety of pathogens, including bacteria, fungi, and atypical pathogens. The diversity of IE manifestations contributes to diagnostic uncertainty, which can lead to missed diagnoses and inappropriate treatment. Diagnosing Coxiella burnetii endocarditis is particularly challenging, as routine blood cultures often yield negative results and echocardiography seldom reveals vegetations. In recent years, the rapid development of sequencing technology provides a new direction for the diagnosis of IE.Case presentationThe present study reports the case of a 57-year-old male patient who was admitted with a diagnosis of severe aortic stenosis. Cardiac computed tomography (CT) angiography revealed a diastolic aortic valve malformation with significant leaflet thickening, calcification and pseudoaneurysm formation, the etiology of which was considered to be due to infectious possibilities. Although clinical suspicion of infective endocarditis was raised, since echocardiogram did not reveal any vegetations and blood cultures were negative, the etiology remained unclear and empirical drug therapy was used. The patient exhibited a severe inflammatory reaction after aortic valve replacement. Samples were sent for pathogenic testing again, including blood cultures and blood nanopore targeted sequencing (NTS). The other tests were all negative, only the NTS detected C. burnetii. After further confirmation, the final diagnosis was C. burnetii infective endocarditis. The patient was subsequently discharged from the hospital after demonstrating improvement with targeted treatment. One month post-discharge, a follow-up examination indicated that he had recovered well.ConclusionThis case study demonstrates the accurate diagnosis and treatment of C. burnetii endocarditis through NTS analysis of blood samples. As a novel technology, NTS may play an important role in the diagnosis of culture-negative infections caused by unidentified pathogens.

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A novel quantitative targeted analysis of X-chromosome inactivation (XCI) using nanopore sequencing.

Publications

A novel quantitative targeted analysis of X-chromosome inactivation (XCI) using nanopore sequencing.

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