To do for a long time RNA-seq analysis, gene expression is also an oral slipped a few years, but it always seems to float on the surface.
Level of understanding of one thing that determines the depth of your mind, just want to do mechanic would not think too much, want to master it must be inquisitive.
Always said gene expression, then what is the expression? We sequenced the gene expression is only a phenotype, is a snapshot of the sample, and the general height and weight like a continuous phenotype similar.
Conventional transcriptome analysis are essentially phenotypic analysis, clustering, pseudotime, DEG, marker , in these analyzes, each gene is independent of the dimension, a static analysis, at this time we are concerned that certain genes functional analysis , such as RET, has made it clear function, it can be explained by another phenotypic expression of this phenotype.
High-throughput sequencing will be a follow-up analysis, one can not be a study of tens of thousands of genes, GO and KEGG analysis came, genes are not independent of each other, concept GO term and a pathway to come. GO and KEGG nature is to regulate the relationship between genes . GO integrates all species, from the perspective of living systems to unify genetic relationship, this relationship is just a collection; KEGG is for a species to define the relationship between genes, this relationship is to have a view of the structure. Must make further in-depth understanding of the principles of GO and KEGG temporarily not in depth. At this point we began to distinguish genotypes , protein coding, non-coding, transcription factors. At this stage we are more concerned with the regulation of the relationship between genes .
Discloses a hierarchical rule and central tube structure of living systems, computer and communications systems are very similar, even if the upstream regulatory base complex then, downstream of the protein is the decisive factor, so that the upstream regulatory stunning so complex, can be downstream of the protein is indeed very stable, indicating that the regulation of the complex is very stable.
The first step in genetic research must be functional genes, followed by the regulatory genes.
Gene function
So how to study the function of a gene it? Reference: # genomic gene function studies of view # "seven trick" and "three tricks" - BioinforCN
Briefly summarize this article:
1. Heaven and Earth and space-time law studies of gene expression to infer function, which is the same investigation and detectives, are indirect reasoning;
2. outcome, the operation is the direct gene, knock out or down or OverExpress, directly explore the function of genes belonging to direct observation;
3. search up and down, because the center level and the rule is a piping system, the downstream is clear, from the gene's DNA, RNA into protein, study together;
4. Ambush perspective argument, do easily observed biological false positive, must demonstrate multi-angle;
5. Other, misexpression, in vitro / vivo.
Do not say the human hs, assuming that you are responsible for a new genomic and gene function studies of species, how do you find all the genes of the species it?
Look genome assembly of any one article can find a solution. Then we look at the latest NG corn Yanjian Bing bar, the Genome Assembly of Line A Tropical Maize inbred has the Provides Insights INTO Structural Variation and Crop Improvement .
Micro letter article: "Nature Genetics" | corn yield-related genes found | tropical maize genome structural variation map and precision successfully constructed, help the genetic improvement of corn
First, the genomic DNA of the assembly, Genome sequencing, assembly and scaffolding, this part of the purely technical, not later estimates are assembled directly to test out the genome;
The second is a genome annotation, Genome annotation, which is part of the part we are most interested in, how to find all the genes in a new species?
A comprehensive strategy combining de novo gene prediction, protein-based homology searches, RNA sequencing (RNA-Seq) and isoform sequencing (Iso-Seq) of nine tissues (Supplementary Table 6) was used to annotate the genes (Supplementary Fig. 7).
The scheme:
1. The gene is a special structure, so long as the DNA sequence can be predicted denovo;
2. The central dogma tells us that DNA, RNA and proteins is a chain, and all measuring RNA-seq and iso-seq can be indirectly introduced gene;
3. Protein sequencing is not yet universal, the homologous protein sequences currently used to reverse thrust;
Such comments out only very general gene annotation, can cover the vast majority of genes, but certainly some special structure can not be commented out.
With sketches, later research function will be a lot easier to do the experiment.
I can only tell you that based on the first two steps of high-throughput sequencing of the genome where genes are, but you could not tell it functions; third step is based on existing knowledge, and do homologous reasoning. So now all knowledge comes from biological experiments, sequencing is only an accelerated aid only.
You can not sequenced, but not without test sequencing research is accelerating catalyst.
Article results:
FINDING METHODS the GENE - the Broad Institute - very comprehensive
Regulation of gene expression / transcription regulatory
Textbook explanation:
- Chromatin structure and chromosomal level, resulting in changes in gene activity;
- The level of transcription regulation ;
- RNA processing level regulation, modification of cut editing degradation;
- After transcription, nuclear transport into the cytoplasm;
- Translational levels;
- Level of protein synthesis;
Two of the most fire-control methods can be used for high-throughput sequencing studies:
- Transcription factors, enhancer, promoter
- Non-coding RNA, lncRNA, miRNA, ceRNA
reference:
Modes of transcriptional regulation
Transcriptional Regulation and Its Misregulation in Disease
Project question:
Now in vivo and in vitro models are mature, RNA-seq cost that everyone can accept, CRISPR technology is mature, KO a gene has become very easy, now in developmental biology, biomedicine and so in doing so: KO a gene to explore the biological processes of interest to what has changed .
Now the question is, after KO phenotype certainly changed, how this phenotype and gene expression and regulation linked to with it ?
This is a general problem, the answer to any good can be used for an in-depth study of genes.
General solution:
Hypothesis testing is the only way to get true knowledge of research, we must first of all be a reasonable assumption, then went looking for evidence to test this hypothesis.
problem:
Building a database program 1. RNA-seq is what? ployA, randomly. There are only caught polyA of MRNA have What are the advantages and disadvantages?
2. The difference between the nucleus and all sequenced?
3. The difference in the length of the gene in the end how much?
4. How is the impact of alternative splicing and isoform protein?
5. KEGG gene which already has a relationship, why we have to study gene regulation?
6. What is the purpose and protein interaction networks is the limitations?
7. The protein is the only decisive factor? Yes, the vast majority of DNA and RNA level change will ultimately change the function of the protein. For example HSCR not form ENS is a complex phenotype, it is certain that some proteins function to perform the disorder.
8. important level of gene expression or gene expression on / off important?
9. The gene is located and how to find? How the protein encoded by the gene is determined?
10. How to understand the relationship between genes, what is the nature of the relationship?
11. How immediately affect gene copy number on gene expression?
12. transposable-element influence on gene expression?
13. The classic structure of genes is what? What is the CDS and UTR? It can be combined with the current mainstream view of gene prediction tools.
14. transcriptional regulation and protein interactions What is the connection and difference?
Typical structure of a mature eukaryotic mRNA (AUG, UAA/UAG/UGA)
Continued ~