Deepening our understanding of meiotic prophase I events in C. elegans by the study of mutants in PROM-1, SMC-3, RRF-3 and live imaging of chromosome end movement

Meiose ist eine spezialisierte Zellteilung, bei der der Chromosomengehalt halbiert wird. Sie ist für die Bildung von haploiden Gameten erforderlich. Während der Prophase der ersten Teilung müssen sich homologe Chromosomenpaare finden, weiters mittels des synaptonemalen Komplexes verbinden und programmierte Doppelstrangbrüche über „Cross-over“ Rekombination repariert werden, um Homologe zu verknüpfen. C. elegans Gonaden rekapitulieren die meiotische Prophase I in zeitlicher und räumlicher Anordnung. Das innere Kernhüllenprotein SUN-1 ist einer der wichtigsten Akteure im Prozess der meiotischen Chromosomen-Paarung. Wir verwendeten SUN-1-Aggregate, um die Bewegung der Chromosomenenden in vivo zu studieren. SUN-1-Aggregate sind sehr dynamisch. Sie kommen zusammen, vereinigen und zerstreuen sich wieder. Anstatt einzelner Chromosomen-Enden, die ihre „Partner“ suchen, haben wir beobachtet, dass Dupletts/ Multipletts und einzelne Chromosomenenden zusammen gebracht werden. Ich analysierte die Anforderungen für die SUN-1 Aggregatbildung, Wildtyp Kinetik der Bewegung und dokumentierte den Einfluss der richtigen Orchestrierung der Meiose und Reparatur von Doppelstrangbrüchen auf das Verhalten der Aggregate. rrf-3 kodiert für eine RNA-Polymerase, beteiligt in endogener RNA Interferenz (RNAi). Genetische Analysen zeigten, dass defekte Spermatogenese für die Unfruchtbarkeit in der rrf-3 Mutante verantwortlich sein könnte. Ich zeigte, dass Paarung der homologen Chromosomen, meiotische Chromosomenachsenmorphogenese und Reperatur von DNA Doppelstrangbrüchen normal waren; nichtsdestotrotz wurden Chromosomenfragmentierung und DNA-Brückenbildung beobachtet während der Spermatogenese. Stattdessen sind Kerne in Spermien von einem Kranz von Tubulin umgeben. „Deep Sequenzierung“ von kleinen RNAs identifizierte Kandidaten Zielgene (Wildtyp und rrf-3 vergleichend), welche von RRF-3 reguliert sein könnten (eine Zusammenarbeit mit Jonathan Jesaja Gent). Cohesin spielt eine wichtige Rolle im Zusammenhalt der Schwesterchromatiden in der Meiose und Mitose. Ich isolierte, klonierte und charakterisierte ein temperaturempfindliches Allel des smc-3 Gens. Ich zeigte, dass die Reparatur der meiotischen Doppelstrangbrüche in dieser Mutante gestört ist, währendessen Kohäsion normal ist. Der primäre Defekt dieser Mutante ist die partielle Ladung von Kohäsinmolekülen, dies führt zu partieller Ladung der Komponenten der Chromosomenachsen und weiters zu defekter Synapse. In meiner Arbeit wurde erstmals ein mutiertes C. elegans smc-3 Allel beschrieben. PROM-1 spielt bei der Orchestrierung von frühen meiotischen Prophase I Ereignissen eine Rolle. Das Fehlen von prom-1 führt zu einem meiotischen Arrest gleich nach dem Eintreten in die Meiose. Ich habe einen Suppressor von prom-1 isoliert mittels eines EMS Mutagenesescreens. Der Suppressor unterdrückt den meiotischen Arrest und führt zu normaler Ladung der Komponenten des synaptonemalen Komplexes. Wiederfunktionierende Paarung im Suppressor resultiert in Ausformung von Bivalenten. Ich habe den Suppressor auf den linken Arme von Chromosome III kartiert. Die hier vorgestellte Arbeit umfasst Einblicke in verschiedene Aspekte der meiotischen Prophase I in C. elegans unter Ausnutzung besonderer Vorteile dieses Modellsystems: der Zugänglichkeit zu genetischer Analyse und Transparenz für in vivo Zeitraffer-Zell Aufnahmen.Meiosis is the specialized cell division that allows halving of the chromosome content and is required for the formation of haploid gametes. During prophase I homologous chromosomes have to pair, synapse via the synaptonemal complex (SC) and repair programmed double strand breaks (DSBs) by cross over recombination to link homologues. C. elegans gonads recapitulate the prophase I of meiosis in a temporal and spatial order. The inner nuclear protein SUN-1 is one of the key players in the process of meiotic chromosome pairing. We used SUN-1 aggregates to monitor chromosome end movement in vivo. SUN-1 aggregates are highly dynamic. They come together, coalesce and disperse. Instead of single chromosome ends looking for their homologous partner, duplets/multiplets and single chromosome ends are brought to meet. I analyzed the requirements for SUN-1 aggregate formation, wild type kinetics and documented the influence of proper orchestration of meiosis and repair of DSBs on aggregate behavior. rrf-3 encodes an RNA-directed RNA polymerase involved in endogenous RNAi. Genetic analysis revealed that defective spermatogenesis accounts for the infertility observed in rrf-3 mutant. I showed that pairing of homologues, meiotic chromosome axes morphogenesis and repair of double strand breaks were normal despite chromosome fragmentation and DNA bridge formation during spermatogenesis. Instead sperm nuclei are surrounded by wreath of tubulin. Deep sequencing analysis of small RNAs comparing wild type and rrf-3 identified candidate targets regulated by RRF-3 (a collaboration with Jonathan Isaiah Gent). Cohesin plays an important role by holding sister chromatids together both in meiosis and mitosis. I isolated, cloned and characterized a temperature sensitive allele of smc-3. I showed that repair of meiotic DSBs is impaired in this mutant whereas cohesion is normal. The primary defect of this mutant is partial loading of the cohesion complex leading to partial loading of chromosome axis components and furthermore defective synapsis. This is the first analysis of an smc-3 disruption in meiosis in C. elegans. PROM-1 is involved in the orchestration of early prophase events. Its absence results in arrest right after meiotic entry. I isolated a suppressor of PROM-1 by EMS mutagenesis. The suppressor allele in the prom-1 deletion mutant suppresses the meiotic arrest, entails the proper loading of SC proteins and successful pairing resulting in formation of bivalents. I mapped the suppressor mutation by single nucleotide polymorphism to the left arm of chromosome III. The work presented here covers insight into various aspects of meiotic prophase I in C. elegans taking advantage of its amenability to forward genetics and its transparency for in vivo time-lapse cell imaging

Characterization of a chromosome rearrangement associated with cardiopathy and autism

Chromosomal rearrangements have been associated with multiple congenital abnormalities, including malformative syndromes and global developmental delay. The aim of this study was identification of candidate genes for a complex phenotype characterized by cardiopathy and autism, identified in an individual with a chromosome translocation t(4;7)(q21.1;p21.2). Since classical and molecular cytogenetic analyses have low resolutions, large-insert whole-genome sequencing (liWGS) was applied for identification and mapping of structural chromosomal alterations. By this approach, the 4q21.1 breakpoint was identified between genomic positions chr4:73,918,924-74,049,529 on 4q13.3, whereas the 7p21.2 breakpoint between chr7:13,184,731-14,536,001 [GRCh38/hg38]; suggesting the occurrence of deletions at both breakpoints. Additionally, a 473Kb deletion on 2p16.3 was also identified in the proband. Nucleotide-level resolution of the breakpoints and familial segregation analysis were carried out by amplification of the junction fragments and Sanger sequencing. At the 4q13.3 breakpoint, the 130Kb deletion erases four genes PF4, PPBP, CXCL5 and CXCL3, whereas at 7p21.2, the 1351Kb deletion removes the entire ETV1 and disrupts DGKB and the long non-coding intergenic (Linc) RNA AC011288.2. Furthermore, at this breakpoint region, genomic array analysis identified in the proband’s father a 742Kb deletion comprising DGKB and ETV but not the LincRNA AC011288.2. The maternally inherited 473Kb deletion on 2p16.3 removes the first 5 exons of NRXN1, a gene associated with Pitt-Hopkins like syndrome (OMIM #614325), susceptibility to schizophrenia and chromosome 2p16.3 deletion (OMIM #614332). Similar deletions have been reported with incomplete penetrance and variable expressivity. Several genes from the 7p21.2 breakpoint region and especially those affected by the deletion, DGKB, ETV1 and LincRNA AC011288.2, have been linked with cognitive, speech, language and auditory disorders. In conclusion, coinheritance of the maternally derived deletion on 2p16.3 and the deletion at the breakpoint of the der(7) on 7p21.2 appear to be the most contributively alterations for the proband’s phenotype. At the time, NRXN1, DGKB, ETV1 and LincRNA AC011288.2 are the most likely genes to be responsible for the proband’s phenotype, being those mainly characterised by cardiopathy and autism

Molecular genetic analysis of paediatric low-grade astrocytoma

The thesis describes the molecular genetic analysis of fifty low-grade paediatric astrocytomas. DNA copy number changes were investigated in paediatric low-grade astrocytomas (WHO grade I and II), using array comparative genomic hybridisation and Affymetrix 250K and 6.0 SNP arrays. A discrete region of DNA copy number gain was identified at chromosome 7q34, primarily although not exclusively in pilocytic astrocytomas of the cerebellum. Further analysis of this region, by PCR and sequencing, demonstrated the presence of gene fusions between KIAA1549 and BRAF. Five KIAA1549- BRAF fusion variants were subsequently identified. A further gene fusion between SRGAP3 and RAF1 was also found in a single tumour with DNA copy number gain at chromosome 3p25. The fusion genes lacked the auto-inhibitory domains of BRAF and RAF1. These were replaced in-frame by N-terminal segments of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. Sequencing confirmed the presence of activating mutations in KRAS and BRAF in three tumours where gene fusions were not identified. Both gene fusions and activating mutations were shown to cause activation of the ERK/MAP kinase pathway by Western blotting. Further sequencing was performed of CDKN2A, PTEN and IDH1/2 to assess the frequency of abnormalities in paediatric low-grade astrocytoma. These genes have previously been found to contain aberrations within adult high-grade astrocytomas. To date, no significant aberrations have been identified in the paediatric astrocytoma samples examined. This confirms previous findings in adult and paediatric astrocytomas, which appear to show distinct molecular changes depending on patient age. Gene fusions or activating mutations were identified in 100% pilocytic astrocytomas studied and were also found in 28% of grade II astrocytomas. These findings highlight the importance of the ERK/MAPK pathway both in the development of paediatric low-grade astrocytomas and as a possible therapeutic target. Gene fusions may provide a means of molecular classification for pilocytic astrocytomas in the future

SPATIAL ORGANIZATION OF LAMIN A/C IN HODGKIN’S LYMPHOMA AND MULTIPLE MYELOMA

Genomic Instability is a complex phenomenon observed both at the DNA and the chromosome level. It is responsible for the generation of mutations and chromosomal rearrangements, which can potentially lead to tumor development. A common genomic instability feature found in some malignant tumors is the shortening of the telomeres, the end of the chromosomes. Shortening of telomeres leads to the formation of anaphase bridges, subsequent breakage and initiation new breakage-bridge-fusion (BFB) cycles. Classical Hodgkin’s lymphoma (cHL) and Multiple Myeloma (MM) are two blood tumor both characterized by genomic instability. cHL is a B-Cell lymphoma comprised of mononuclear Hodgkin cells (H) and bi- to multi-nucleated Reed-Sternberg (RS) cells, both characterized by numerical (nCIN) and structural chromosomal instability (sCIN). Transition from H to RS cells is correlated with aberrant number of mitotic spindles, aberrant 3D telomere organization, increase in the number of telomere aggregates and telomere loss. Advanced shortening of telomeres leads disruption of 3D interaction of the telomere with TRF2, a member of the shelterin proteins responsible for the protection of the chromosome ends from the DNA damage repair system. MM, an incurable plasma cell disorder, is also characterized by CIN, microsatellite instability (MSI), and telomere attrition. Lamin A/C, a nuclear envelope protein and component of the nuclear matrix, is important for the assembly of the mitotic spindles and for the maintenance of the 3D telomeres architecture by binding the shelterin protein TRF2. Due to lamin A/C involvement in the maintenance of genomic stability, our goal was to investigate lamin A/C 3D spatial organization in cHL and MM, and understand whether the localized uncapping of TRF2 from telomeres leads to their detachment from the nuclear matrix component lamin A/C causing genomic instability. Western blot analysis and qRT-PCR analysis revealed that H and RS are characterized by an overall overexpression of lamin A/C when compared to the B-lymphocytes control. Three dimensional (3D) fluorescent microscopy in cHL and MM cells revealed that the regular homogeneous lamin A/C pattern identified in normal activated B-lymphocytes, was replaced by an aberrant lamin A/C 3D spatial distribution characterized by the presence of internal lamin A/C structures. Different lamin A/C patterns where observed where the internal lamin A/C sub-divided the nucleus in to two or multiple compartments, suggesting a possible involvement of lamin A/C in the multi-nucleation process. To investigate lamin A/C involvement in the multinucleation process and transition from H to RS, the downregulation of lamin A/C with siRNA was performed. Downregulation of lamin A/C affected the transition from H to RS cells, as the number of RS cells found after lamin silencing was decreased. Telomere 3D structure and nuclear organization were also affected by the deregulation of lamin A/C. H and RS cells were also characterized by unbinding of telomere-TRF2-lamin A/C when compared to the healthy control. Lack of co-localization of telomere-TRF2-lamin A/C confirms lamin A/C involvement in genomic instability, and suggests that localized absence of TRF2 could be causal to the genome remodeling in cHL

Electron tomography of meiotic spindles in males of the trioecious nematode Auanema rhodensis

The nematode Auanema rhodensis has recently been established as a new model organism. A. rhodensis is characterized by the simultaneous existence of three phenotypical sexes (males, females and hermaphrodites; called trioecy), skewed sex ratios which do not follow Mendel's laws, and variant segregation patterns according to sex and type of gametogenesis. Recently, A. rhodensis has been used to study the possible mechanisms for sex determination in three-sexed species and the variability of basic processes during sexual reproduction including meiotic divisions. During male meiosis, a diploid primary spermatocyte undergoes two consecutive divisions to form four haploid spermatids. Surprisingly, male meiosis in A. rhodensis results in two functional and two nonfunctional spermatids depending on the presence of an X-chromatid. A. rhodensis males exhibit a set of paired autosomes and one single X-chromosome, like males of the wellestablished nematode model organism Caenorhabditis elegans. In contrast to C. elegans, however, the X-chromosome in A. rhodensis divides precociously into its sister chromatids during the first meiotic division followed by a lagging X-chromatid and its uneven distribution during meiosis II. Additionally, the second meiotic divi-sion within this species is characterized by an asymmetric organelle distribution and a spindle structure reminiscent of a monopolar spindle. In this study, serial section electron tomography was used to analyse the ultrastructure of the microtubule skeleton in spermatocytes of A. rhodensis. The analysis of spermiogenesis using electron tomography posed some key advantages compared to standard transmission electron microscopy. First, the microtubule (MT) network could be studied in detail including spindle formation, organization of spindle poles, rearrangement of MTs, and inter-action between MTs and chromosomes. Second, the number and shape of chromosomes could be visualized. And third, the morphology of organelles could be observed at high resolution, and different organelles as well as their distribution pattern could be distinguished and quantified. This study provides highresolution 3D information about male meiosis in A. rhodensis. The results of this thesis confirm the complexity of the male meiotic program and the promi-nent position of the X-chromatid in meiosis II in this organism. Like previous light microscopic studies, electron tomography supports the hypothesis of an X-chromatid-dependent distribution of cellular organelles such as fibrousbody membranousorganelles (FB-MOs) and mitochondria. Furthermore, the formation of an asymmetric spindle could be observed with progressing anaphase II and might be associated with the X-chromatid distribution. Additionally, the analysis of the number of chromosome-associating MTs and their association character gives new insights into possible chromosome segregation mechanisms. Finally, significant differences to the male meiotic program in C. elegans have been identified. For the first time, the MT network in A rhodensis spermatocytes of different division stages has been observed in detail, and several different analyses could be done, including an analysis of the length distribution of MTs in the spindles. Because this ultrastructural analysis is based on fixed samples, live-cell imaging should be performed in the future to gain further information on the chromosome dynamics in this species.Der Fadenwurm Auanema rhodensis hat sich in den letzten Jahren mehr und mehr zu einem neuen Modellorganismus entwickelt. Typisch für A. rhodensis ist das zeitgleiche Vor-kommen dreier phänotypischer Geschlechter (Männchen, Weibchen und Hermaphroditen; die sogenannte Triözie) und deren zahlenmäßig ungleiches Verhältnis zueinander, welches sich nicht durch Mendelsche Regeln erklären lässt. Ebenfalls auffällig sind nach Geschlecht und Gametogenese (Oogenese oder Spermatogenese) abweichende chromosomale Segregationsmuster. Unlängst hat A. rhodensis zu Erkenntnissen über die Geschlechtsdeterminierung in dreigeschlechtlichen Arten und die Varianz grundlegender Prozesse in der Meiose beigetragen. Während der männlichen Meiose (Spermatogenese) teilt sich eine diploide primäre Vorläuferzelle (primäre Spermatozyte) in zwei aufeinanderfolgenden Teilungen in insgesamt vier haploide Spermatiden. Bei A. rhodensis führt die Spermatogenese ungewöhnlicher-weise zu zwei funktionalen und zwei nicht-funktionalen Spermatiden, wobei die Funktionsfähigkeit vom Auftreten eines X-Chromatids abhängt. Männchen von A. rhodensis besitzen, ähnlich wie im Modellorganismus Caenorhabditis elegans, eine Reihe gepaarter Autosomen sowie ein einzelnes X-Chromosom. Im Gegensatz zu C. elegans teilt sich das ungepaarte X-Chromosom in A. rhodensis vorzeitig schon während der ersten meiotischen Teilung in seine Chromatiden, wodurch es zu einer verzögerten und ungleichen Verteilung des X-Chromatids während der zweiten meiotischen Teilung kommt. Diese zweite meiotische Teilung bei A. rhodensis ist außerdem durch eine asymmetrische Verteilung der Organellen und Mikrotubuli gekennzeichnet, letztere ähneln einer monopolaren Spindel. In dieser Arbeit wurde die Methode der seriellen Elektronentomographie genutzt, um die Ultrastruktur der Mikrotubuli in meiotischen Spindeln in Spermatozyten von A. rhodensis zu untersuchen. Zum einen wurden mittels Elektronentomographie das Netzwerk der Mikrotubuli und die Spindelorganisation, die Struktur der Spindelpole sowie die Interaktion zwischen Mikrotubli und Chromosomen drei-dimensional (3D) analysiert. Zum anderen wurde die Form der Chromosomen und die Morphologie und Verteilung der verschiedenen Organellen quantitativ erfasst. Somit stellt diese Studie hochauflösende 3D-Information über den Ablauf der männlichen Meiose in A. rhodensis zur Verfügung und bestätigt damit die Komplexität der männlichen Meiose und die zentrale Rolle des X-Chromatids während der zweiten meiotischen Teilung in diesem Organismus. Basierend auf vorangegangenen lichtmikroskopischen Experimenten an fixierten Proben unterstützt die Elektronentomographie die Hypothese einer vom X-Chromatid abhängigen Verteilung zellulärer Organellen wie spermienspezifischer FB-MOs oder Mitochondrien während der zweiten meiotischen Teilung. Außerdem konnte die Ausbildung einer asymmetrischen Spindel beobachtet werden, welche ebenfalls mit der ungleichen Verteilung des X-Chromatids in Zusammenhang stehen könnte. Eine zusätzliche Analyse chromosomenassoziierter Mikrotubuli brachte erste Erkenntnisse über mögliche zugrundeliegende Mechanismen der Chromosomensegregation. Die Ergebnisse dieser Arbeit konnten mit ähnlichen Untersuchungen in C. elegans verglichen und Unterschiede herausgearbeitet werden. Zum ersten Mal wurden hier meiotische Spindeln unterschiedlicher Teilungsstadien in 3D untersucht und unterschiedliche quantitative Analysen zur Längenverteilung der Mikrotubuli durchgeführt. Da alle hier gewonnenen Ultrastrukturdaten auf fixierten Proben basieren, sollte eine Betrachtung einer transgenen Wurmlinie mit Fluoreszenzmarkern mittels live-cell imaging auf diese Ultrastrukturanalyse folgen

Image Processing and Simulation Toolboxes of Microscopy Images of Bacterial Cells

Recent advances in microscopy imaging technology have allowed the characterization of the dynamics of cellular processes at the single-cell and single-molecule level. Particularly in bacterial cell studies, and using the E. coli as a case study, these techniques have been used to detect and track internal cell structures such as the Nucleoid and the Cell Wall and fluorescently tagged molecular aggregates such as FtsZ proteins, Min system proteins, inclusion bodies and all the different types of RNA molecules. These studies have been performed with using multi-modal, multi-process, time-lapse microscopy, producing both morphological and functional images. To facilitate the finding of relationships between cellular processes, from small-scale, such as gene expression, to large-scale, such as cell division, an image processing toolbox was implemented with several automatic and/or manual features such as, cell segmentation and tracking, intra-modal and intra-modal image registration, as well as the detection, counting and characterization of several cellular components. Two segmentation algorithms of cellular component were implemented, the first one based on the Gaussian Distribution and the second based on Thresholding and morphological structuring functions. These algorithms were used to perform the segmentation of Nucleoids and to identify the different stages of FtsZ Ring formation (allied with the use of machine learning algorithms), which allowed to understand how the temperature influences the physical properties of the Nucleoid and correlated those properties with the exclusion of protein aggregates from the center of the cell. Another study used the segmentation algorithms to study how the temperature affects the formation of the FtsZ Ring. The validation of the developed image processing methods and techniques has been based on benchmark databases manually produced and curated by experts. When dealing with thousands of cells and hundreds of images, these manually generated datasets can become the biggest cost in a research project. To expedite these studies in terms of time and lower the cost of the manual labour, an image simulation was implemented to generate realistic artificial images. The proposed image simulation toolbox can generate biologically inspired objects that mimic the spatial and temporal organization of bacterial cells and their processes, such as cell growth and division and cell motility, and cell morphology (shape, size and cluster organization). The image simulation toolbox was shown to be useful in the validation of three cell tracking algorithms: Simple Nearest-Neighbour, Nearest-Neighbour with Morphology and DBSCAN cluster identification algorithm. It was shown that the Simple Nearest-Neighbour still performed with great reliability when simulating objects with small velocities, while the other algorithms performed better for higher velocities and when there were larger clusters present

Aneuploidy in Health and Disease

Aneuploidy means any karyotype that is not euploid, anything that stands outside the norm. Two particular characteristics make the research of aneuploidy challenging. First, it is often hard to distinguish what is a cause and what is a consequence. Secondly, aneuploidy is often associated with a persistent defect in maintenance of genome stability. Thus, working with aneuploid, unstable cells means analyzing an ever changing creature and capturing the features that persist. In the book Aneuploidy in Health and Disease we summarize the recent advances in understanding the causes and consequences of aneuploidy and its link to human pathologies

차세대 염기서열 분석 장비로 생성한 메타지놈 데이터 분석을 위한 최적의 생물정보학 시스템 개발

학위논문 (박사)-- 서울대학교 대학원 : 협동과정 생물정보학전공, 2014. 2. 천종식.Metagenome is total DNA directly extracted from environment, and the purpose of metagenomics is to reveal the function of the metagenome as well as the taxonomic structure in the metagenome. There are two analysis approaches for metagenomics, namely amplicon based approach and random shotgun based approach. Both approaches require large scale sequencing reads which could not be satisfied through Sanger sequencing. However, high throughput sequencing of reads at relatively low cost by Next Generation Sequencing (NGS) technologies meets the requirement of metagenomics. In addition, the advent of NGS technologies gave rise to the development of bioinformatic algorithms necessary for processing this large and complex sequencing data. Consequently, the large amount of sequencing data obtained from NGS and corresponding proper bioinformatic algorithms facilitated the metagenomics to become essential tool for microbiology. However, limitations incurred by NGS sequencing errors, short read length, and lack of analysis system still hinder accurate metagenome analysis. Therefore, evaluation of currently used NGS error handling algorithms and development of systematic pipeline with more efficient algorithms are required to improve the accuracy of analysis. In this study, bioinformatic pipelines were constructed for both metagenome analysis approaches. The pipelines were dedicated to improve the accuracy of the final end result by minimizing the effect of errors and short read length. For the amplicon based metagenomics, two different analysis pipelines were developed for both 454 pyrosequencing and Illumina MiSeq. During the construction of 454 pyrosequencing pipeline, new error handling algorithm was developed to treat homo-polymer and PCR errors. Upon completion of the pipeline construction, household microbial community was analyzed using 454 pyrosequencing data as a case study. As for Illumina MiSeq data, the most appropriate sequencing conditions and sequencing target region were settled. Paired end merging programs were evaluated and correlation of the sequencing errors and quality was studied to correct the errors within 3 overlap regions. Novel iterative consensus clustering method was developed to correct the errors occurring ubiquitously in a single read. For shotgun metagenomics approach, bioinformatic analysis system for Illumina MiSeq paired end data was constructed. Unlike the targeted amplicon sequencing reads, most of the shotgun sequencing reads are not mergedthus short reads are used for both functional and taxonomical profiling. However, a short read has less information than longer contigs, so the use of short reads is likely to cause biased characterization of the metagenome. Therefore, the development of analysis system did focus on creating longer contigs by means of mapping and de novo assembly. For raw read mapping, a dynamic mapping genome set construction method was developed. A list of mapping genomes was selected from the taxonomic profile inferred from the ribosomal RNA profiles. The genome sequence of the selected genomes were downloaded from Ezbiocloud. By mapping raw reads to the genome sequences, the longer contigs can be obtained in case of the relatively simple metagenome such as fecal matter. However in case of the complex metagenomes such as soil sample, both mapping and de novo assembly did not perform properly due to a lack of sequencing coverage and numerousity of uncultured microorganisms in the metagenome. In addition to the pipeline construction, visualization tools were also developed to display resultant taxonomic and functional profile at the same time. Newly developed JAVA-based standalone sequence alignment editing application was named as EzEditor. As both, conserved functional coding sequences and 16S rRNA gene have been used copiously in bacterial molecular phylogenetics, the codon-based sequence alignment editing functions are required for the coding genes. EzEditor provides simultaneous DNA and protein sequence alignment editing interface which enables us with the robust sequence alignment for both protein and rRNA sequences. EzEditor can be applied to various molecular sequence involved analysis not only as a basic sequence editor but also for phylogenetic application.ABSTRACT I TABLE OF CONTENTS IV ABBREVIATIONS VI FIGURE LIST VII TABLE LIST XII Chapter 1 General Introduction 1 1.1 Bioinformatics 2 1.2 Next Generation Sequencing 5 1.3 Metagenomics 11 1.4 Objectives of This Study 21 Chapter 2 Amplicon-based Metagenome Analysis Systems 23 2.1 Introduction 24 2.2 Analysis System for 454 Pyrosequencing 35 2.2.1 Methods 36 2.2.2 Results 39 2.3 Analysis System for Illumina MiSeq 60 2.3.1 Methods 62 2.3.2 Results 68 2.4 Summary and Discussion 93 Chapter 3 Shotgun-based Metagenome Analysis System 99 3.1 Introduction 100 3.1.1 Tools for Metagenomics 101 3.2 Methods 118 3.3 Results 125 3.4 Summary and Discussion 165 Chapter 4 EzEditor: A versatile Molecular Sequence Editor for Both Ribosomal RNA and Protein Coding Genes 169 4.1 Overview 170 4.2 Features of EzEditor 172 4.2.1 Algorithms and Models Implemented in EzEditor 177 4.2.2 Miscellaneous Functions 178 4.3 Summary and Discussion 181 Conclusions 183 References 187 APPENDIX I. Estimated Diversity Index of Household Microbiome 217 국문 초록 (Abstract in Korean) 221Docto