Telomere and Proximal Sequence Analysis Using High-Throughput Sequencing Reads

The telomere is a specialized simple sequence repeat found at the end of all linear chromosomes. It acts as a substrate for telomere binding factors that in coordination with other interacting elements form what is known as the shelterin complex to protect the end of the chromosome from the DNA damage repair machinery. The telomere shortens with each cell division, and once critically short is no longer able to perform this role. Short dysfunctional telomeres result in cellular senescence, apoptosis, or genome instability. Telomere length is regulated by many factors including cis-acting elements in the proximal sequence which is known as the subtelomere. The Riethman lab played a pivotal role in generating the reference sequence of the subtelomere in both the human and mouse genomes, providing an essential resource for this work. Short high throughput sequencing (HTS) reads generated from the simple repeat containing telomere or the segmental duplication rich subtelomere cannot be aligned to a reference genome uniquely. They are filtered and excluded from many HTS analysis methods. A ChIP-Seq analysis pipeline was developed to incorporate these multimapping reads to study DNA-protein interactions in the subtelomere. This pipeline was employed to search for factors regulating the expression TERRA, an essential long non-coding RNA, and to better characterize their transcription start sites. ChIP-seq analysis in the human subtelomere found colocalization of CTCF and Cohesin directly adjacent to the telomere and throughout the subtelomere specific repeats. Follow up functional studies showed this binding regulated TERRA transcription at these sites. Extending these analyses in the mouse genome showed very different patterns of CTCF and cohesin binding, with no evidence of binding at apparent sites of TERRA transcription. Mouse subtelomere sequence analysis showed the co-occurence of two repeats at sites of putative TERRA expression, MurSatRep1 and MMSAT4, one of which was previously shown to be expressed in lincRNAs. The Telomere Analysis from SEquencing Reads(TASER) pipeline was developed to capture telomere information from HTS data sets and used to investigate telomere changes that occur in prostate cancer. TASER analysis of 53 paired prostate tumor and normal samples revealed an overall decrease in telomere length in tumor samples relative to matched paired normal tissue, especially sequence containing the exact canonical telomere repeat. Multimapping reads contain important information, that when used properly, help elucidate understanding of telomere biology, cancer biology, and genome regulation and stability

Development and application of mammalian molecular cytogenetic tools for genome reconstruction, evolution and reproductive screening

Chromosomal analysis enables a genome-wide overview of an organism, it can provide information when used to study cellular function, the taxonomic relationship between divergent species and disease phenotypes. Consequently, chromosomal analysis is used to identify chromosomal rearrangements in an individual, which can be associated with disease and/or reproductive complications, or within a population, which is associated with speciation and reproductive isolation. The techniques used to examine the chromosomes of an organism have improved considerably over the past four decades. Observations were traditionally achieved through the production of Giemsa stained chromosomes which permitted banding analysis, therefore enabling the detection of differences in chromosome morphology and number, to more specific, molecular cytogenetic approaches (fluorescence in situ hybridisation - FISH) which can be used to identify sub-microscopic differences. Today, genome sequencing facilitates genome-wide analysis at a higher resolution than previously possible; sequence information can be used in a multitude of ways, including identification of specific mutations which result in disease, investigating homologous segments between divergent species and for ascertaining potential drug targets. However, without a physical genetic map it is now apparent that by themselves genome sequence assemblies fail to provide sufficient information regarding certain biological questions, in particular genome organisation throughout times of mammalian evolution. However, it is now apparent that map-based chromosome-level assemblies are required for deeper analysis of the genome. With this in mind, the purpose of this work was to extend upon, and develop efficient cytogenetic tools to screen for chromosomal rearrangements in mammalian species, in the context evolutionarily events and to examine chromosomal rearrangements that manifest as fertility problems in a range of agricultural and zoological animals. Using traditional karyotyping techniques, Ducos et al (2007) demonstrated that the translocation incidence rate was 0.47% in unproven boars. In this work, a large number of boars (>1000) were analysed using a FISH-based screening device, whereby 13 unique chromosomal translocations were detected, resulting in an incidence rate of 1%. Therefore, the results in this work demonstrate that the incidence rate is under reported in the current literature. Before this work, karyotype analysis was the only technique used to identify chromosomal rearrangements in cattle. As a consequence of the success observed in pigs, a FISH-based device was developed to screen for chromosomal translocations in cattle. Using this technology, heterozygous and homozygous 1;29 translocations were identified, and an unreported 12;23 reciprocal translocation

Fusion genes in breast cancer

Fusion genes caused by chromosomal rearrangements are a common and important feature in haematological malignancies, but have until recently been seen as unimportant in epithelial cancers. The discovery of recurrent fusion genes in prostate and lung cancer suggests that fusion genes may play an important role in epithelial carcinogenesis, and that they have been previously under-reported due to the difficulties of cytogenetic analysis of solid tumours. In particular, breast cancers often have complex, highly rearranged karyotypes which have proved difficult to analyse using classical cytogenetic techniques. The aim of this project was to search for fusion genes in breast cancer by using high-resolution mapping of chromosome rearrangements in breast cancer cell lines. Mapping the chromosome rearrangements was initially done using high-resolution DNA microarrays and fluorescence in- situ hybridisation, but moved to high-throughput sequencing as it became available. Interesting candidate genes identified from the mapped chromosome rearrangements were investigated on a larger set of cell lines and primary tumours. The complete karyotypes of two breast cancer cell lines were constructed using a combination of microarrays, fluorescence microscopy, and high-throughput sequencing. A number of potential fusion genes were identified in these two cell lines. Although no expressed fusion genes were found, the complete karyotypes gave insight into the number and mechanisms of chromosome rearrangement in breast cancer, and identified interesting candidate genes which may be of importance in tumourigenesis. Two genes which were fused in other breast cancer cell lines, BCAS3 and ODZ4, were disrupted by chromosome rearrangements and identified as interesting candidate genes in tumorigenesis. A bioinformatic pipeline to process high-throughput sequencing data was set up and validated, and shown to more accurately predict fusion genes than other methods, and can be used to investigate further cell lines and tumours for recurrent fusion genes. The pipeline was used to analyse data from 3 other breast cancer cell lines and predict chromosomal rearrangements and fusion genes, several of which were found to be expressed. Of the fusions predicted in the cell line ZR-75-30, 7 expressed fusion genes were identified, and may have functional significance in breast cancer.This work was supported by a grant from Breast Cancer Campaign

The role of complex chromosome translocations in leukemia

Resumo A tumorigénese é um processo de transformação celular que se desenrola tipicamente em várias etapas. Os diferentes níveis de evolução tumoral resultam da acumulação sucessiva de mutações genéticas numa célula normal que lhe conferem uma vantagem selectiva no respectivo meio tecidular. As mutações podem manifestar-se sob a forma de alterações nucleotídicas pontuais ao nível da sequência de DNA, levando a uma desregulação da função proteíca ou à formação de proteínas não-funcionais, ou através de alterações cromossómicas numéricas ou estruturais. Na leucemia, por exemplo, os genes híbridos que resultam de translocações cromossómicas desempenham um importante papel no processo tumorigénico. Estes genes são transcritos sob a forma de um RNA mensageiro de fusão, o qual é traduzido numa proteína híbrida com função oncogénica. Frequentemente, os subtipos de doença leucémica estão associados com translocações cromossómicas que envolvem 2 pontos de quebra recorrentes e específicos. É disto exemplo a leucemia mielóide crónica, em que uma translocação recíproca entre os cromossomas 9 e 22 conduz à formação de um gene de fusão BCR-ABL1. Em diferentes subtipos de doença, existe também uma pequena proporção de casos que apresenta translocações cromossómicas complexas, que envolvem um ou mais pontos de quebra adicionais em outras localizações genómicas além das que estão implicadas na formação dos genes de fusão. Por vezes, os pontos de quebra estão também associados a delecções extensas de material genético que se pensa terem uma função importante na tumorigénese. No entanto, o papel destas regiões genómicas no desenvolvimento tumoral não tem sido um motivo recorrente de estudo. Neste contexto, o objectivo desta dissertação foi o de determinar o potencial papel tumorigénico de alterações génicas adicionais ocorridas nos pontos de quebra de translocações cromossómicas complexas. Para a prossecução do objectivo proposto, foram estudados 5 rearranjos cromossómicos distintos associados com diferentes tipos de doença hematológica maligna, nomeadamente a leucemia linfoblástica aguda de células B (2 casos), leucemia mielóide aguda, neoplasma mieloproliferativo e síndrome mielodisplásico/neoplasma ieloproliferativo, não classificável. O mapeamento dos pontos de quebra foi efectuado utilizando a hibridação fluorescente in situ e diferentes metodologias de biologia molecular, tendo como base a informação inicial da análise citogenética. Em casos seleccionados, o papel dos novos genes candidatos foi avaliado in vitro utilizando modelos de linhas celulares, nomeadamente no que respeita às funções de controlo da proliferação celular e de regulação transcricional. De entre os 5 casos estudados, quatro deles evidenciaram translocações complexas envolvendo 3 cromossomas, nomeadamente t(12;21;5)(p13;q22;q13), t(12;6;15)(p13;p24~25;q22), t(9;11;19)(p22;q23;p13) e t(X;20;16)(p11;q13;q23). No caso remanescente, foi observada uma translocação dicêntrica dic(9;12)(p11;p11) acompanhada de delecções extensas em ambos os pontos de quebra. Nos casos com t(12;21;5) e t(9;11;19) as translocações estavam associadas com a presença de genes de fusão recorrentes, nomeadamente TV6(12p13)-RUNX1(21q22) e TLL(11q23)-MLLT3(9p22), indicando que se tratavam de rearranjos complexos das translocações t(12;21) e t(9;11) associadas com a leucemia linfoblástica aguda de células B e a leucemia mielóide aguda, respectivamente. O papel dos pontos de quebra adicionais foi estudado em detalhe no caso com t(9;11;19). Através da metodologia de long distance inverse-polymerase chain reaction, foram identificados os pontos de quebra na sequência de DNA dos 3 cromossomas envolvidos na translocação. Além dos pontos de quebra nos genes MLL e MLLT3, foi observado que o local de quebra no cromossoma 19 interrompeu a sequência de um novo gene, designado CCDC94,conduzindo à sua haplo-insuficiência nas células com t(9;11;19). Através de ensaios de reverse transcription-polymerase chain reaction verificámos que o gene CCDC94 é expresso ubiquitariamente em tecidos humanos normais. A análise informática da sequência prevista da proteína CCDC94 indicou uma elevada identidade de aminoácidos com a proteína cwf16, envolvida na regulação do ciclo celular da levedura Schizosaccharomyces pombe. Através da clonagem do DNA complementar de CCDC94 em vectores de expressão, e após a transfecção destes em culturas de linhas celulares in vitro, observámos que este gene codifica uma proteína de localização exclusivamente nuclear. A expressão ectópica da proteína CCDC94 diminuiu a progressão do ciclo celular e a proliferação das células em cultura. Inversamente, a supressão do transcrito do gene CCDC94 através de interferência de RNA conduziu a um aumento significativo da proliferação celular, confirmando que CCDC94 regula negativamente a proliferação e a progressão do ciclo celular. Estes resultados mostram que os pontos de quebra adicionais, presentes em translocações cromossómicas complexas em leucemia, podem resultar na haplo-insuficiência de genes controladores dos mecanismos proliferativos, cooperando desta forma com a acção das proteínas de fusão para proporcionar ao clone leucémico uma proliferação celular descontrolada. Nos restantes 3 casos estudados não foram identificados genes de fusão. Ao invés, todos aqueles apresentaram delecções de extensão variável associadas com os pontos de quebra cromossómicos. No caso com t(12;6;15), identificámos uma delecção de 1.2 megabases de DNA na banda 12p13 que resultou na eliminação de 9 genes incluindo ETV6 e CDKN1B. O gene ETV6 codifica um factor de transcrição que é essencial para a formação das diferentes linhagens hematopoiéticas na medula óssea, enquanto CDKN1B é traduzido numa proteína responsável por bloquear a entrada das células na fase G1 do ciclo celular e,consequentemente, por travar a proliferação celular. Neste contexto, os resultados obtidos indicam que a perda simultânea de ETV6 e de CDKN1B, através de uma translocação cromossómica complexa, constituiu uma acção cooperativa na leucemogénese. A mesma noção pode aplicar-se ao caso com dic(9;12), no qual pelo menos 2 genes que codificam para factores de transcrição importantes na linhagem hematopoiética, PAX5 no cromossoma 9 e ETV6 no cromossoma 12, estavam deleccionados como resultado do rearranjo cromossómico. Dado que o factor de transcrição PAX5 regula negativamente a expressão do gene FLT3, que desempenha uma função pró-proliferativa, é expectável que a haplo-insuficiência de PAX5 no caso com dic(9;12) terá tido como consequência uma elevação dos níveis de expressão de FLT3, contribuindo deste modo para uma proliferação celular aumentada. A t(X;20;16) foi identificada num doente com trombocitémia essencial (TE), uma doença que está intimamente relacionada com alterações de vias intracelulares reguladas por citocinas. Neste caso, através da utilização de um array genómico, identificámos a presença de pequenas delecções associadas com os pontos de quebra nos cromossomas 16 e 20. No cromossoma 16 apenas um gene, MAF, estava deleccionado, enquanto no cromossoma 20 a delecção tinha abrangido 3 genes. Dos genes deleccionados, dois deles, NFATC2 (20q13) e MAF (16q23), codificam proteínas que operam como reguladores transcricionais de citocinas hematopoiéticas. Dado que NFATC2 se localiza numa região que constitui um alvo frequente de delecções em neoplasmas ieloproliferativos, incluindo a trombocitémia essencial,efectuámos um estudo detalhado do papel deste gene na proliferação megacariocítica e na regulação da expressão de uma citocina hematopoiética (GM-CSF), implicada na maturação das diferentes linhagens mielóides. Utilizando um modelo de linha celular de trombocitémia essencial, verificámos que a supressão do transcrito do gene NFATC2 in vitro, por interferência de RNA, estava associada com um aumento da proliferação celular. Em concordância, o bloqueio da activação da proteína NFATC2 através de um inibidor específico da sua interacção com a calcineurina, conduziu a um aumento da proliferação celular in vitro. Utilizando a PCR quantitativa em tempo real, detectou-se um aumento da produção do RNA de GM-CSF em ambos os ensaios celulares, indicando que o factor de transcrição NFATC2 pode regular negativamente a expressão de GM-CSF em células de trombocitémia essencial. No geral, estes resultados mostram que a redução dos níveis fisiológicos do transcrito NFATC2, ou a redução da respectiva actividade proteica, estão relacionados com a proliferação de megacariocitos através do aumento da produção de GM-CSF. De acordo com estes resultados, verificámos que as células dos doentes com TE apresentam níveis mais baixos do transcrito NFATC2 do que a população normal. Dado que o factor de transcrição MAF desempenha igualmente um papel como regular transcricional de citocinas, é plausível que a haplo-insuficiência dos genes NFATC2 e MAF, resultante do rearranjo cromossómico complexo t(X;20;16), teve um efeito cooperativo importante na patogénese da trombocitémia essencial através da alteração do padrão normal de expressão das citocinas hematopoiéticas. Em síntese, efectuámos nesta dissertação um estudo citogenético de 4 translocações cromossómicas complexas incluindo t(12;21;5), t(12;6;15), t(9;11;19) e t(X;20;16), e de uma translocação dicêntrica dic(9;12), associadas com diferentes neoplasmas hematológicos. Em casos seleccionados efectuámos também um estudo molecular detalhado das regiões dos pontos de quebra. Esta análise permitiu-nos identificar 2 genes, CCDC94 no cromossoma 19 e NFATC2 no cromossoma 20, cuja haplo-insuficiência pode promover o aumento da proliferação celular das células leucémicas. A partir destes estudos podem ser retiradas 2 noções principais: (i) Os pontos de quebra adicionais, que ocorrem em translocações complexas associadas com a formação de genes de fusão, podem ter como consequência a desregulação de genes controladores da proliferação celular (e.g., CCDC94); (ii) As translocações complexas caracterizadas pela ausência de genes de fusão recorrentes poderão estar preferencialmente associadas com a presença de delecções, envolvendo um ou mais genes, nos pontos de quebra; nestas situações, serão necessários pelo menos 2 genes com funções celulares semelhantes (e.g., NFATC2 e MAF) ou complementares (e.g., ETV6 e CDKN1B) para, quando deleccionados, promoverem de forma cooperativa a leucemogénese. Nestes termos, o modelo de alterações genéticas sequenciais que caracteriza o desenvolvimento do cancro pode ser substituído por um modelo em que vários genes-alvo são simultaneamente desregulados pela formação de uma translocação cromossómica complexa, evitando deste modo a necessidade de ocorrência de alterações genéticas subsequentes.----------------------ABSTRACT: Tumourigenesis is a multistep process which results from the accumulation of successive genetic mutations in a normal cell. In leukemia for instance, recurrent translocations play a part in this process by generating fusion genes which lead to the production of hybrid proteins with an oncogenic role. However, a minor subset of chromosomal translocations referred to as complex or variant involves extra breakpoints at variable genome locations in addition to those implicated in the formation of fusion genes. We aimed to describe in this work the role, if any, of genes located at extra breakpoint locations or which are affected by breakpoint-adjacent deletions through the study of 5 leukemia patients.Two of the patients presented with TV6(12p13)-RUNX1(21q22) and MLL(11q23)- MLLT3(9p22) fusion genes as a result of a t(12;21;5) and a t(9;11;19), respectively. Detailed molecular characterization of the extra breakpoint at chromosome 19 in the latter case revealed that a novel ubiquitously expressed gene, CCDC94, with a potential role in cell cycle regulation, was disrupted by the breakpoint. We demonstrated using in vitro cellular assays that this gene codifies for a nuclear protein which negatively regulates cell cycle progression. These data shows that extra breakpoint locations of complex translocations may result in haplo-insufficiency of critical proliferation genes, thereby cooperating with the generation of hybrid proteins to provide unrestrained cell proliferation. In the other 3 patients there were reakpoint-associated deletions which precluded the formation of putative fusion genes. In a case with a t(12;6;15) we characterized a deletion at 12p13 which eliminated ETV6 and 8 other genes including CDKN1B. These findings indicate that concomitant loss of ETV6 and CDKN1B, which encodes a cyclin-dependent kinase inhibitor responsible for blocking entry of cells into the G1 phase of the cell cycle, acted cooperatively to promote leukemogenic proliferation. The same notion applied to a case with a dic(9;12) in which 2 genes encoding hematopoietic transcription factors - ETV6 and PAX5 (9p13)- were deleted as a result of breakpoint-adjacent deletions. Similarly, we found that 2 transcription factor genes involved in the regulation of cytokine expression, NFATC2 (20q13) and MAF (16q23), were involved in deletions contiguous to the breakpoints in a patient with a t(X;20;16). In vitro suppression of NFATC2 mRNA or inhibiton of NFATC2 protein activity enhanced cell proliferation as a result of an increase in the production of a myeloid-lineage stimulating hematopoietic cytokine, GM-CSF. These results suggest that haplo-insufficiency of NFATC2 and MAF genes had a cooperative effect in inducing cell proliferation as a result of a disregulation of cytokine production. Two main conclusions may be drawn from our studies: (i) In complex translocations associated with the production of fusion genes, additional breakpoints may cooperate in tumourigenesis by targeting genes that control cell proliferation; (ii) In complex translocations associated with small breakpoint-adjacent deletions, at least 2 genes with similar or complementary functions need to be deregulated to promote tumourigenesis

Identification and mapping of loci controlling viability in Vitis vinifera crosses

The present research work is part of the NOVABREED project, funded by the European Research Council (ERC). The focus of NOVABREED is the concept of pan-genome as a comprehensive representation of a species genome: its main aim is to characterise the dispensable portion that arises as a consequence of structural variation, and its contribution to the intra-specific genetic and phenotypic variation (Morgante, De Paoli, & Radovic, 2007). The focus of the present work is on the Vitis vinifera genome. The goal of the present research is to understand the genetic bases of inbreeding depression in grapevine through the identification of loci controlling viability and survival and their relationship with structural variation. Vitis vinifera is a very variable species with high levels of both nucleotide diversity and structural variation (Gabriele Magris, PhD thesis, 2016). Cultivated varieties are highly heterozygous (Jaillon et al., 2007) and are expected to carry a large mutational load that is reflected in rather severe inbreeding depression observed upon crossing of related individuals or selfing. We set out to identify loci that are responsible for inbreeding depression both individually as well as a consequence of epistatic interactions. Through the analysis of segregation distortion, defined as deviation of segregation ratio from the expected Mendelian ratio, we explored progenies segregation pattern with the goal of isolating causative mutations of the distortion. In order to characterize segregation distortion in Vitis vinifera, progenies obtained from self-crosses of six varieties and from one out-cross were genotyped using a Genotyping-by-Sequencing approach. The technique used, known as double digest Restriction Site Associated DNA Sequencing (ddRAD-seq) (Peterson, Weber, Kay, Fisher, & Hoekstra, 2012), subsamples the genome at homologous locations among individuals by coupling double restriction enzyme digestion to a selection of genomic fragments by size, allowing fine-scale control of the fraction of regions represented in the final library. SNP genotyping, by means of the software Stacks (Catchen, Hohenlohe, Bassham, Amores, & Cresko, 2013), allowed the identification of several regions of single locus distortion in each progeny assessed. Progenies deriving from self-fertilization of the six cultivars Cabernet franc, Pinot Noir, Primitivo, Rkatsiteli, Sangiovese and Schiava Grossa, showed distortion in 12 different loci. Progeny of the out-cross between Schiava Grossa and Rkatsiteli did not show any locus of segregation distortion. Overall, ten loci of distortion revealed lethal effects, whereas two loci revealed severe deleterious effects. Among loci with lethal effect, seven showed the complete absence of a homozygous genotype, corresponding to the action of recessive alleles; two showed the action of partially dominant alleles and one showed nearly complete dominance of the lethal allele. Deleterious loci exhibited recessive and overdominant allelic effect on distortion, respectively. In three different varieties, chromosome 8 was revealed to harbour non overlapping loci of distortion with different allelic effects. Analysis of selected progenies over two vegetative seasons showed that five loci caused embryo or seedling lethality in early stages of growth, while two loci exerted their lethal effects on growth only after the first wintering. Fine mapping of the distorted regions allowed to narrow down the causal locus to less than 34 Kbp, in the best case. Genotype data of the progeny of Rkatsiteli self-cross and of the progeny of Schiava Grossa and Rkatsiteli out-cross was used to build genetic linkage maps of the parental varieties. The three genetic maps were used to analyse recombination frequency along the genome of Vitis vinifera. To identify structural variants (SVs), single nucleotide polymorphisms (SNPs) and small insertion-deletion polymorphisms (INDELs) contributing to the genetic load in the grapevine genome and leading to segregation distortion in the progenies of selfing, candidate loci were screened for mutations affecting genes. Haplotype phasing of alleles allowed to identify mutations belonging to the haplotype that generated segregation distortion in the progenies of selfing. Furthermore, expression of genes affected by such variation was evaluated in a panel of six varieties in three different tissues (leaf, berry and tendril). The analysis of two-loci segregation distortion was also performed in order to identify epistatic interactions. Fisher\u2019s test of independence revealed one strong signal of interaction between loci on chromosome 1 and chromosome 11 in the variety Rkatsiteli, evidenced also by the pseudo-linkage signal in the genetic map. Further evidences showed that the interaction was actually due to a balanced translocation, which was validated through a PCR-based assay. In order to evaluate gene expression differences between the haplotype carrying the translocation and the normal haplotype in Rkatsiteli, we performed allele specific expression (ASE) analysis for each of the three tissues. Interestingly, the analysis revealed no significant difference in the allele-specific expression profile in the tissues. In order to detect the balanced translocation in other grapevine varieties, a panel of 196 cultivars was tested, revealing that three varieties - Alexandroouli, Mtsvane Kachuri and Gorula - carried the rearrangement. All four varieties originated from Georgia and belonged to the Proles pontica, although not all the varieties of this Proles showed the translocation. This suggests that Georgian varieties are distinct and genetically variable from western one and that translocation could be an ancient event never imported to the West and Central Europe varieties (Proles occidentalis). Our study showed that self-fertilization of Vitis vinifera cultivars lead to high levels of segregation distortion in the progenies due to the presence of unfavourable alleles in genes. Future studies will be oriented to the characterization of the identified genes. Furthermore, we generated a fine-scale map of recombination frequency along the genome of Vitis vinifera. Lastly, we detected and validated a balanced translocation involving chromosome 1 and chromosome 11 in the variety Rkatsiteli and we found the chromosomal rearrangement in other three grapevine varietie

Understanding The Complexity of Human Structural Genomic Variation Through Multiple Whole Genome Sequencing Platforms

Genomic structural variants (SVs) are major sources of genome diversity and closely related to human health, as indicated by numerous studies. In spite of the recent advances in sequencing technology and discovery methodology, there are still considerable amounts of variants in the genome that are partially or completely misinterpreted. This thesis has mainly focused on comprehensively interpreting the structural variants in human genomes by accurately defining the locations and formats of variants with the application of different sequencing platforms. To accomplish this goal, I developed a randomized iterative approach to define all types of SVs, which has shown superior performance in accurately defining complex variants. Next, I built a recurrence based validation pipeline to systematically validate SVs with long read sequences. I conclude with a systematic integration of SVs in multiple individuals discovered by various short read based detecting algorithms, with supportive evidence from orthogonal technologies, which presents to date the most comprehensive SV map in the human genome and the best current technologies allow us to do.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138462/1/xuefzhao_1.pd

Pan-cancer analysis of whole genomes identifies driver rearrangements promoted by LINE-1 retrotransposition

About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage–fusion–bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors