A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.
Human strongyloidiasis a soil-transmitted infection caused by Strongyloides stercoralis is one of the most neglected amongst the so-called Neglected Tropical Diseases (NTDs). S. stercoralis is a nematode, which is distributed worldwide; it has been estimated that it could affect millions of people, mainly in tropical and subtropical endemic regions. The difficulties of diagnosis lead to infection rates being underreported. Asymptomatic patients have chronic infections that can lead to severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. Strongyloidiasis can easily be misdiagnosed because conventional faecal-based techniques lack of sensitivity for the morphological identification of infective larvae in faeces. None of the currently used molecular methods have used urine samples as an alternative to faecal samples for diagnosing strongyloidiasis. This study was thus aimed at comparing, for the first time, the use of a new loop-mediated isothermal amplification (LAMP) molecular assay (Strong-LAMP) to traditional methods on patients’ urine samples. Twenty-four urine samples were taken from patients included in a study involving two Spanish hospitals for strongyloidiasis screening using parasitological and serological tests. Strongyloides larvae were found in 11 patients’ faecal samples, thereby ascertaining that they had the disease. Other patients had high antibody titres but no larvae were found in their faeces. All urine samples were analysed by PCR and Strong-LAMP assay. No amplification occurred when using PCR. Strong-LAMP led to detecting S. stercoralis DNA in urine samples from patients having previously confirmed strongyloidiasis by parasitological tests and/or a suspicion of being infected by serological ones. The Strong-LAMP assay is a useful molecular tool for research regarding strongyloidiasis in human urine samples. After further validation, the Strong-LAMP assay could also be used for complementary and effective diagnosis of strongyloidiasis in a clinical setting.
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