RNA-seq based Transcriptome analysis on domestic animals under various experimental design

Abstract

Today, next-generation sequencing (NGS) technology can produce billions of nucleotides sequences in a single run. In addition, hundreds and thousands of papers in various research fields are published using NGS technology. The NGS technology is now the most powerful tool for the biological science and evolutionary science, and it produces more information than the whole information of the previous studies. RNA sequencing (RNA-seq) is a recent technique that appeared presently after next-generation sequencing (NGS) was invented. In the study of gene expression profiling, Transcriptome sequencing is most appropriate because it enables a profile of the whole transcriptome. A full view of a cellular transcriptional profile at a given biological point and remarkably improve the power of RNA detection methods are provided by whole-transcriptome sequencing. In the application of NGS approach for RNA, several studies were successfully implemented. In the near future, every researcher will use NGS for RNA such as RNA-seq routinely, but transcriptome analysis doesnt come easy to them. Therefore, this thesis is mainly about researches using RNA-seq and additional DNA re-sequencing with NGS, from complex data that including expression information and additional evolutionary information of genes. In chapter 1, the general background of NGS was summarized. The history of sequencing technologies and the classification of NGS methods were presented, more detail, the separation of NGS methods such as genomic and transcriptomic, which was listed. The character of RNA-seq was summarized. The history of sequencing and gene expression were presented, and comparison between RNA-seq and previous technologies, and an overview of RNA-seq analysis was presented. Evolution of domestic animals (Horse, Pig and Chicken) was introduced. .In chapter 2, by using RNA-seq data in a Jeju native pigs and a Berkshire pig in three different tissues (fat, liver and muscle), significantly change of gene expression pattern of response to breed in each tissue was investigated. Jeju native pigs (JNP) have been adapted to an exotic natural environmental niche. They have been known to be resistant disease and have a good meat quality because of higher tenderness, juiciness, redness and brightness than those of Western breeds. In order to understand the molecular mechanisms of JNP specific phenotype, here I conducted comparative transcriptomics study using RNA-seq technology. I compared transcriptome between JNP and Berkshire in three different tissues (fat, liver and muscle). I identified differential expressed genes (DEGs) of each tissue between the two breeds. Among the DEGs, I found that 26 genes were related to meat quality and body growth. Among those genes, MPZ, AADAT, IGFN1 and MYBPH were up-regulated in JNP. Therefore, I suggest that JNP has different gene expression profile which related to meat quality and body growth compared to Berkshire. In chapter 3, by using RNA-seq data in 9 chicken broiler of 3 different calcium intake condition, significantly change of gene expression pattern of response to calcium–stress in kidney tissue was investigated. Chicken (Gallus gallus) was first domesticated from a wild form called a red jungle fowl that still runs wild in most of Southeast Asia. After then, the grey jungle fowl (G. sonneratii) was hybridized, which was occurred probably about 8,000 years ago, and domestic chickens have been selected to have ideal economic traits such as a meat breed and a laying breed. Among these breeds, a meat breed such as a broiler is the most distributed in poultry industry. In addition, calcium is essential for normal cellular function and blood coagulation. However, it has a decisive effect on the hypocalcemia and the hypercalcemia if calcium intake was less or more than adequate calcium intake, which is related to body weight gain and stress such as hypertension. So, I experimented about the body weight gain and feed intake from 4 chicken broilers per calcium intakes (0.8, 1.0 and 1.2percent) and generated RNA-seq data from 10 broilers for gene expression profiling. As a result, I identified differentially expressed genes (DEGs) using cufflinks (128 DEGs between 0.8 and 1.0 percent, 141 DEGs between 0.8 and 1.2 percent and 103 DEGs between 1.0 and 1.2 percent), and also 12 DEGs were identified by edgeR. I identified that these DEGs were related to hypertension and blood pressure through the KEGG pathway enrichment, the co-occurrence and the protein/protein interaction (PPI) network analysis. Next, seven DEGs that were randomly chosen were validated by quantitative real-time PCR (qRT-PCR). In summary, the objective of this study was to investigate the influence of increasing calcium intake in broilers kidney. Therefore, I suggested that higher calcium intakes than adequate amount in broilers can cause the reduced body weight gain that was related to stress-induced disease such as hypertension. In chapter 4, previous studies of horse RNA-seq were performed by mapping sequence reads to the reference genome during transcriptome analysis. However in this study, I focused on two main ideas. First, differentially expressed genes (DEGs) were identified by de novo–based analysis (DBA) in RNA-seq data from six Thoroughbreds before and after exercise, here-after referred to as de novo unique differentially expressed genes (DUDEG). Second, by integrating both conventional DEGs and genes identified as being selected for during domestication of Thoroughbred and Jeju pony from whole genome re-sequencing (WGS) data, we give a new concept to the definition of DEG. I identified 1,034 and 567 DUDEGs in skeletal muscle and blood, respectively. DUDEGs in skeletal muscle were significantly related to exercise-induced stress biological process gene ontology (BP-GO) terms: immune system process

response to stimulus

and, death and a KEGG pathways: JAK-STAT signaling pathway

MAPK signaling pathway

regulation of actin cytoskeleton

and, p53 signaling pathway. In addition, I found TIMELESS, EIF4A3 and ZNF592 in blood and CHMP4C and FOXO3 in skeletal muscle, to be in common between DUDEGs and selected genes identified by evolutionary statistics such as FST and Cross Population Extended Haplotype Homozygosity (XP-EHH). Moreover, in Thoroughbreds, three out of five genes (CHMP4C, EIF4A3 and FOXO3) related to exercise response showed relatively low nucleotide diversity compared to the Jeju pony. DUDEGs are not only conceptually new DEGs that cannot be attained from reference-based analysis (RBA) but also supports previous RBA results related to exercise in Thoroughbred. In summary, three exercise related genes which were selected for during domestication in the evolutionary history of Thoroughbred were identified as conceptually new DEGs in this study. In chapter 5, in this study, I aim to identity that differentially expressed isoforms (DEIs), differential splicing and alternative splicing event by using the published Thoroughbred racing horse RNA-seq data between before and after exercise, because previous studies didnt researched that carefully and without researches about alternative splicing event in Thoroughbred racing horses. I used g/--GTF-guide option in Cufflinks program, because I want to find the all reference transcripts as well as any novel genes, isoform and splicing. As results, In DEIs, the number of DEI in blood and skeletal muscle were 67 and 1,133 respectively. Among them, novel DEIs were 37 in blood, 378 in skeletal muscle. In addition, I identified 7 (6 up-regulated and 1 down-regulated) DEIs in blood and 56 (45 up-regulated and 11 down-regulated) DEIs in skeletal muscle. Among them, in blood, 3 isoforms such as HSPA8 (heat shock 70 kDa protein 8 gene), RhoB (Rho-related GTP-binding protein) and SOCS3 (suppressor of cytokine signaling 3 mRNA) (up-regulated) in blood and 5 isoforms such as AMPD2 (AMP Deaminase Isoform L), ICAM1 (intercellular adhesion molecule 1), MMP-1 (Matrix metalloproteinase-1), MXD1 (MAX Dimerization Protein 1) and TET2 in skeletal muscle were revealed that related to exercise-induces. Moreover, we identified 4 (4 up-regulated) significant differential splicing such as BLZF1, ITGB6, KDM5C and ZNF207 gene in skeletal muscle. Most of these genes were included a litter-related exercise-induce stress with alternative splicing. Conclusively, we classified and identified the alternative splicing events in blood and skeletal muscle in six Thoroughbreds racing horses before and after exercise. As a result, we identified that exon skipping/inclusion (ESI) type is the most common of alternative splicing event, this is the identical result such as human and yeast but the different result as pig with alternative 3 splicing (A3) Through these studies, diverse applications, of the transcriptome analysis considering the experimental design and purpose, was successfully demonstrated in RNA-seq data or additional re-sequencing data derived from NGS. By using data acquired from RNA-seq or additional re-sequencing technology, lots of biological and evolutionary meaning could be achieved. Given these results, I suggest that researchers in transcriptome study field will employ the suitable transcriptome analysis corresponding to their experimental design and purpose.