3.6. Mutation detection

By analyzing the transcriptome sequencing data, you can obtain sequence information for all transcripts of the sample, including all SNP and Indel mutation types on the transcript. The RNA molecules generated by transcription may undergo various modifications before translation, such as the RNA editing process of A-to-I, thereby further increasing the complexity of the transcriptome. Analyzing the mutation information in the transcript can capture the modification process of the gene from DNA to RNA transcription, thereby exploring the complex regulatory mechanism in the transcription process.

4. The Application of RNA-seq

4.1. Determining gene expression patterns

RNA-seq technology makes it faster and easier to study gene expression in the entire transcriptome. For species with or without a reference genome, different analysis strategies can be used to efficiently obtain cells or cells in a specific state. All transcript information in the tissue, and then study the differences in gene expression patterns between different tissues or developmental stages. In addition, with the accumulation of transcriptome sequencing data, various transcriptome databases have been established. The accumulation of this information will be used for further research on the types and expression levels of transcripts under specific physiological conditions, the interaction between transcripts, and transcriptional regulation etc.

4.2. Using to discover new transcripts

Transcriptome sequencing based on NGS technology can obtain complete transcript sequences through a splicing algorithm, but for genes with multiple alternative splicing forms, it is still difficult to identify between different transcripts. Single-molecule sequencing technology is applied to transcriptome sequencing. You can take advantage of the longer read length of the sequence to obtain the complete transcript sequence directly. There is no need to assemble the NGS short read to accurately obtain different alternative spliced transcript sequences and the amount of expression.

4.3. Regulation mechanism of non-coding RNA

Non-coding RNA cannot form protein products, but it plays an important regulatory function that cannot be ignored in biological processes. In recent years, a large number of ncRNAs have been found in various cells and tissues through NGS technology and RNA-seq. Their types and expression levels often reflect species characteristics and specific physiological states. Especially in the research of animal and plant breeding, disease resistance and environmental adaptability, the role of ncRNA is getting more and more attention.

4.4. Single cell transcriptome

The study of transcripts at the level of a single cell can provide an in-depth analysis of the heterogeneity between cells, thereby analyzing the physiological characteristics of different types of cells. For a specific organization to study its function, it is necessary to understand and distinguish its cell type composition. Different cell types may play specific physiological functions. scRNA-seq has become a solution to deeply study the difference of transcripts of different types of cells and the combination between cells. At present, there are a variety of methods that can achieve single cell isolation and conduct scRNA-seq research and are widely used in the study of tumor cell transcriptome.

Transcriptome sequencing technology can determine the structure of messenger RNA (mRNA) and non-coding RNAs (ncRNA) sequences and transcribed genes, and quantitatively and dynamically express the mode conditions of each transcript under different biology. With the further development of sequencing technology, it provides a variety of new solutions for transcriptome positioning and quantitative research. In general, the current RNA-seq is still mainly based on second-generation sequencing, and RNA-seq based on third-generation sequencing technology has become an important direction of transcriptomics research. The current high-throughput sequencing technology needs to go through the library construction process. First, a large number of RNA molecules are enriched and converted into a cDNA sequencing library. During the library construction process, fragments of the cDNA molecules are connected to sequencing primer adapters and sample labeling sequences. The development of RNA-seq technology provides an effective means for studying the relationship between transcription regulatory networks and traits from the overall transcription level.


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