Project description:Tuberous sclerosis complex (TSC) is a relatively common autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms, caused by loss-of-function mutation of either TSC1 or TSC2. Target-capture full-length double-stranded cDNA sequencing using long-read sequencer Nanopore (Nanopore Long-read Target Sequencing) revealed that the various kinds of the TSC1 and TSC2 full-length transcripts and the novel intron retention transcripts of TSC2 in TSC patient. Our results indicate that the Nanopore Long-read Target Sequencing is useful for the detection of mutations and confers information on the full-length alternative splicing transcripts for the genetic diagnosis.
Project description:Analysis and understanding of transcript functions is greatly helped by knowing the full-length sequence of individual RNAs. New long-read sequencing devices such as Oxford Nanopore and Pacbio have the potential to sequence full-length transcripts, but standard methods lack the ability to capture true RNA 5’ ends and selects for poly-adenylated (pA+) transcripts. We present a method that, by utilizing cap-trapping and 3’ end adapter ligation, can sequence transcripts from the exact 5’ end to 3’ end regardless of whether they are poly-adenylated, with no need for ribosomal RNA depletion. We show that the method can faithfully detect 5’ ends, splice junctions and 3’ ends, has high reproducibility between runs and gene expression estimates from the method correlate well with short-read sequencing methods. We also demonstrate that the method can detect and sequence full-length pA- RNAs, including lncRNAs, promoter upstream transcripts (PROMPTs) and enhancer RNAs. TLDR-seq is therefore useful for the characterization of diverse capped RNA species.
Project description:Analysis and understanding of transcript functions is greatly helped by knowing the full-length sequence of individual RNAs. New long-read sequencing devices such as Oxford Nanopore and Pacbio have the potential to sequence full-length transcripts, but standard methods lack the ability to capture true RNA 5’ ends and selects for poly-adenylated (pA+) transcripts. We present a method that, by utilizing cap-trapping and 3’ end adapter ligation, can sequence transcripts from the exact 5’ end to 3’ end regardless of whether they are poly-adenylated, with no need for ribosomal RNA depletion. We show that the method can faithfully detect 5’ ends, splice junctions and 3’ ends, has high reproducibility between runs and gene expression estimates from the method correlate well with short-read sequencing methods. We also demonstrate that the method can detect and sequence full-length pA- RNAs, including lncRNAs, promoter upstream transcripts (PROMPTs) and enhancer RNAs. TLDR-seq is therefore useful for the characterization of diverse capped RNA species.