ONT-TlrRNA-seq

Second – generation transcriptome sequencing cannot provide the results of quantitative analysis for multiple transcripts of a gene respectively, because this technology is not suitable for such a situation. A single read of second – generation short sequencing cannot span the full – length transcript. It is necessary to use software to splice short reads to obtain transcripts, and then evaluate the expression level. There may be problems of splicing errors or incompleteness, so the complete transcript cannot be accurately obtained, and the source of reads of multiple transcripts of a gene cannot be distinguished. There is a phenomenon of multiple alignments of short reads in highly conserved regions with high similarity among different genes, which leads to inaccurate or biased quantitative expression at the transcript or even gene level. In contrast, Nanopore third – generation full – length sequencing does not require breaking and splicing transcripts. One read can span the full – length transcript, and the multiple alignment rate is low, so it can accurately obtain the respective expression information of multiple full – length transcripts of a gene. From second – generation transcriptome to Nanopore third – generation full – length transcriptome, platform upgrading and technological innovation have been realized, which can solve the problems that cannot be solved by second – generation sequencing.

However, compared with second – generation sequencing technology, the length of each read in third – generation sequencing technology is significantly longer. When the amount of sequencing data is the same, the number of reads generated by third – generation sequencing will be significantly less than that by second – generation sequencing. For some transcripts with low expression levels, this requires extremely high sequencing depth to achieve effective detection. For example, when performing saturation detection of the human transcriptome, more than 30 Gb of sequencing data is required. Such high requirements will inevitably greatly increase the cost of third – generation sequencing and bring a greater economic burden to research work. Targeted sequencing technology enables researchers to screen out gene loci of interest, thereby reducing sequencing costs and workload, so as to obtain data with high coverage in target regions.

Oxford Nanopore Technologies Targeted Long – Read Sequencing (ONT – TlrRNA – seq) is to amplify the full – length region of the transcript of interest by PCR, and then directly sequence the amplicons.

The core advantage of ONT – TlrRNA – seq technology is that it can perform deep sequencing for specific transcripts without comprehensively sequencing the entire transcriptome. This method not only significantly improves the sequencing efficiency, but also enables researchers to focus more on transcripts directly related to diseases or specific biological processes.

Through ONT – TlrRNA – seq technology, researchers can quickly identify the expression of transcripts of key genes, providing strong support for subsequent transcript function research and disease diagnosis. In addition, ONT – TlrRNA – seq technology is also widely favored because of its high flexibility and simple operation, and has become an important technology in the current field of transcript detection.