(The) use of next generation sequencing to decipher metagenome of domestic animals

다국어 초록 (Multilingual Abstract)

This study investigated metagenomic characteristics of microbial communities in the domesticated poultry through the combination of next generation sequencing (NGS) and bioinformatic pipelines. Among various kinds of commercial animals, this study placed its focus on the bovine rumen, chicken feces and goat rumen fluid, respectively. In more detail, structure of microbial population in the various samples of rumen (bovine/goat) and feces (chicken) was analyzed and then, its phylogeny and specific gene such as cellulase was identified. Sequencing the genomes of rumen microbes, determining the role of the genes and identifying its potential applications are the great deal for researchers to understand the microbiology of the rumen. Chicken feces have generally been known to be closely associated with contamination of poultry and health safety. Goats have unique habits, which include feeding on unconventional tree leaves. Thus, goats are expected to host distinct bacterial communities with cellulose-degrading enzyme activity in their rumen.
In cahpter1, basic background and necessity were reviewed the series of worked in this doctoral dissertation, which showed its pivotal role of microbiomes in the commercial animal in various respects. Furthermore, metagenomics and genetics/genomics can provide a significant clue to these microbial population.
In chapter 2, the microbial community structure of rumen solid and rumen liquid of cattle rumen was analyzed using high-throughput pyrosequencing of 16S rRNA gene PCR amplicons and a subsequent bioinformatics pipeline. A 16S rRNA gene clone library identified abundant communities belonging to specific bacterial groups in the rumen. The diversity results suggested that the specific bacterial groups was found in both samples with a slight difference. Bacteroidetes, Firmicutes and Fibrobacteria were present in higher concentrations in rumen solid than in rumen liquid, indicating their major role in the degradation of plant fiber. Other groups identified include Proteobacteria, which are responsible for playing a greater role in rumen metabolism; Mollicutes class of Firmicutes, which metabolizes imported sugars to short chain fatty acids; and Prevotellacea, which are crucial for the breakdown of proteins. These biological function of identified microbial groups mentioned above have also coincided with other previous studies.
In chapter 3, fecal microbial community of chicken was quantitatively analyzed using next generation sequencing (NGS) techniques and bioinformatic analyses with metagenomic tools such as MOTHUR, MEGA6 etc. during a relatively short growth time of 35 days. The diversity of microbial community at the genus level increased during the five week growth period (from 30 to 87 identified genera). Despite the diversity, only a few dominant bacteria groups (over 80%) were identified in each fecal sample, which were completely different from each other. These results suggested that chicken fecal microbiome is a dynamic system with a differentiated population structure under a restricted number of higher taxa.
In chapter 4, protein domains with cellulase activity in goat rumen microbes were investigated using Illumina sequencing and bioinformatic analyses with metagenomic tools such as METAIDBA, HMMER and Interproscan etc. After the complete genome of the goat rumen microbe was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 439 genera was analyzed, among which Prevotella and Butyrivibrio were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4) were obtained through blast searches using the enzyme.dat file provided by the NCBI database. 28 protein domains with cellulase activity were identified using the HMMER package. Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, celluase, and Glyco hydro 10 were present in bacteria with strong cellulase activities. Furthermore, correlation plot clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. Recent studies clearly reported that intestinal microbome was closely correlated with the traits of host such as obesity and growth. Therefore, it can be easily expected that analysis of intestinal microbial structure in commercial animals can provide an insight to the livestock industry in terms of fiber digestion and growth.

목차 (Table of Contents)

• ABSTRACT..................................................I
• CONTENT..................................................V
• LIST OF TABLES........................................VI
• LIST OF FIGURES......................................VIII
• LIST OF APPENDIX.....................................XI
• CHAPTER 1. LITERATURE REVIEW................1
• 1.1 SEQUENCING TECHNIQUES....................2
• 1.2 METAGENOMICS..................................28
• 1.3 METAGENOMIC APPROACH IN DOMESTIC ANIMALS...................................................38
• CHAPTER 2. MICROBIAL COMMUNITY STRUCTURES OF RUMEN SOLID AND LIQUID IN CATTLE.........44
• 2.1 ABSTRACT...........................................45
• 2.2 INTRODUCTION....................................46
• 2.3 MATERIALS AND METHODS..................49
• 2.4 RESULTS.............................................58
• 2.5 DISCUSSION........................................69
• CHAPTER 3. ANALYSIS OF BACTERIA COMMUNITY CHANGE IN CHICKEN FECES DURING THE REARING PERIOD USING NEXT GENERATION SEQUENCING............................................71
• 3.1 ABSTRACT...........................................72
• 3.2 INTRODUCTION....................................73
• 3.3 MATERIALS AND METHODS..................75
• 3.4 RESULT...............................................80
• 3.5 DISCUSSION........................................87
• CHAPTER 4. METAGENOME ANALYSIS OF PROTEIN DOMAIN COLLOCATION WITHIN CELLULOSE GENES OF GOAT RUMEN MICROBES..........................91
• 4.1 ABSTRACT..........................................92
• 4.2 INTRODUCTION...................................94
• 4.3 MATERIALS AND METHODS.................96
• 4.4 RESULTS...........................................101
• 4.5 DISCUSSION......................................110
• GENERAL DISCUSSION............................112
• REFERENCES.........................................114
• APPENDIX..............................................138
• 요약(국문초록)........................................155