Case 1: RNA-seq identification of endosperm differentiation related regulatory modules by laser capture microdissection of maize components
Research background:
Endosperm is a nutritive component in angiosperms for embryo development or seed germination. In cereals, endosperm is the main source of food, feed and industrial raw materials in the world. However, the gene network that regulates the differentiation of endosperm cells is largely unknown.
Research content:
In order to study the gene network during endosperm development, we first studied the mRNA expression of 5 cell types of endosperm: 1 embryo and 4 maternal components.
Research results:
Co-expression modules related to cell types were identified through gene co-expression network analysis, and some imprinted genes and sequence-activated genes that have been studied were enriched in the modules. Through in-depth analysis of the modules related to the endosperm transport layer, we identified the key regulatory gene MRP-1 of the module, which regulates the differentiation and function of the endosperm transport layer. Through comparison, it was found that there were different expression patterns between the components of grain and maternal line, and there was a higher correlation between the aleurone layer of embryo and endosperm.
These studies provide a high-resolution network set for the gene activity of different components of maize kernel, and contribute to the study of the regulation module of the main endosperm cell type differentiation.

Figure 1. PCA analysis of gene expression of different cell components Figure 2 Regulation network of endosperm transport layer related modules
Case 2 Improved genetic diagnosis of Mendelian disease by transcriptome sequencing
Research background:
The emergence of WES and WGS has greatly accelerated our ability to identify mutated genes. The mutations of these known and new pathogenic genes can explain many Mendelian diseases. Although these technologies are the mainstream methods for analyzing Mendelian diseases, their success rate in detecting these pathogenic mutations is still low, and the main challenge is that the ability of WES and WGS to detect gene mutations greatly exceeds our ability to explain the functional and clinical impact of these mutations.
Research content:
One way to further improve our understanding of these genetic variations is to integrate functional genomic information, such as RNA-seq. The cDNA analysis of single case and single gene of Mendelian disease patients has been proved to be effective, and RNA-seq has been used to detect some pathogenic mutations, which have been identified by DNA sequencing before. In this study, researchers extracted RNA from damaged muscle tissue in patients with primary muscular diseases (including myopathy and muscular dystrophy) and performed RNA-seq. Among these cases, 13 have been previously diagnosed to carry mutations that affect transcription, such as functional deletion mutations or splice site mutations, to verify the accuracy of RNA-seq in detecting abnormal transcription events. The remaining 50 undiagnosed patient cohorts include cases with mutations or strong candidate genes predicted by DNA sequencing that are most likely to affect splicing, and cases with candidate genes not screened in genetic diagnosis.
Research results:
Gene expression and homologous isomers vary greatly among different tissues. This shows that for many diseases, sequencing disease-related tissues is very important to correctly explain genetic variation. The expression of the most common pathogenic genes in most muscle diseases is not high in blood and fibroblasts, which indicates that RNA-seq using tissues easily obtained from blood or fibroblasts may not be sufficient to detect the transcription abnormalities in some genes. Therefore, the researchers chose to extract RNA from the original muscle tissue biopsy samples. Muscle biopsy is a routine analysis process for diagnosis and evaluation of patients with muscle disease.

Figure 1: Some abnormal splicing events identified in patients Figure 2: Recurrent splicing site mutations in four patients with collagen dystrophy
reference:
1. Zhan J, Thakare D, Ma C, et al. RNA sequencing of laser-capture microdissected compartments of the maize kernel identifies regulatory modules associated with endosperm cell differentiation[J]. Plant Cell, 2015, 27(3):513-531.
2.Beryl B,Jamie L,Taru T,et al.Improving genetic diagnosis in Mendelian disease with transcriptome sequencing[J]. ence translational medicine, 2017, 9(386):eaal5209.