If you want to use your existing data to find genes/pathways/networks related to diseases, then go out and go straight to differential expression analysis.
First look at the sequencing peaks. If the color spectrum of the result shows that the peak pattern is sharp and single, and the code corresponding to the base is consistent, it can be judged that the sequence can be used. If there are abnormal phenomena such as double peaks and signal interruption, it needs to be re-prepared or cloned before sequencing.
Generally, the multiple of the target gene relative to the standard gene can be determined by fluorescence quantitative PCR, and the number of the target gene can be known by selecting a certain benchmark. But sometimes, due to the small amount of DNA samples, the second generation high-throughput sequencing method is used to detect chromosome multiples.
After sequencing the gene sequence, it is generally necessary to compare the sequence to see the difference between it and the target sequence. The commonly used software is DNAman, and vectorVI of invitrogen is also good. In addition, it can also be compared directly on the internet, and it is suggested that the blasting of NCBI website can be carried out directly on the internet.
Cloning into plasmid vector and centralized sequencing [9, 10]. The final result of sAGE is obtained by computer statistics, and the abundance of gene expression is judged and calculated according to the frequency of a tag.