RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta tese será disponibilizado somente a partir de 23/08/2024. SÃO PAULO STATE UNIVERSITY “JÚLIO DE MESQUITA FILHO” BIO SIENCES INSTITUTE BOTUCATU POSTGRADUATE PROGRAM IN BIOLOGICAL SCIENCES (GENETICS) MARYAM JEHANGIR A NOVEL APPROACH TO ASSEMBLE THE COMPLEX B CHROMOSOME USING A COMBINATION OF MODERN GENOMICS TECHNOLOGIES Botucatu, SP 2022 Maryam Jehangir A NOVEL APPROACH TO ASSEMBLE THE COMPLEX B CHROMOSOME USING A COMBINATION OF MODERN GENOMICS TECHNOLOGIES Thesis submitted to the Postgraduate Program in Biological Sciences (Genetics) of the Institute of Bio sciences of Botucatu from the State Universityof São Paulo “Júlio de Mesquita Filho” for achievement of the Ph.D. in Biological Sciences (Genetics). Advisor: Prof. Dr. Cesar Martins Botucatu, SP 2022 Acknowledgment I acknowledge all, including the institutions, funding agencies, mentors, committee members, colleagues and my family who in one way or another contributed in the completion of this PhD thesis. I gratefully acknowledge the Post-Graduate Program in Biological Sciences (Genetics) of the University São Paulo State University, for the opportunity to expand my knowledge and making it possible for me to accomplish Ph.D. as international student. This work would not have been possible without the financial support of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The project of my PhD was funded by CAPES (process number: 88882.433287/2019.01) for my doctoral scholarship. Additionally, I was awarded a fellowship for research internship abroad (Ph.D. sandwich program, process number: 201042/2020-7). Both CAPES and CNPq are acknowledged for supporting my studies and research. My special and heartily thanks to my supervisor, Prof. Dr. Cesar Martins who encouraged and directed me in this work and significantly contributed to my academic training. During my academic tenure of Ph.D., Prof. Cesar gave me intellectual freedom in my work, supporting my attendance at various conferences, engaging me in new ideas, and demanding a high quality of work in all my endeavors. His challenges brought this work towards a completion. It is with his supervision that this work was accomplished. For any faults I take full responsibility. I am also deeply thankful to Assoc. Prof. Dr. Kornsorn Srikulnath who accepted me in his laboratory for the accomplishment of my research internship abroad, allowing me to experience advanced training in an international environment. I also acknowledge the Department of Genetics, Kasetsart University, Thailand for hosting me during the 2 years training, research and academic activities. Additionally, I would like to thank my committee members for their interest and contributions in my work. This thesis was accomplished with the help and support of my fellow lab mates and collaborators, Adauto Lima Cardoso, Jordana Oliveira, Ivan Wolf, Luiz Bovolenta, Camila Moreira and my lab colleagues from Animal Genomics and Bioresource center, Kasetsart University. I also thank my family who encouraged me and prayed for me throughout the time, and my husband, Farhan Ahmad, for him continued support, encouragement and collaboration in my research. Also not forgetting my daughter, Inshirah, her cute smile gives me more courage. Maryam Jehangir Table of Contents 1 INTRODUCTION.............................................................................................................................1 1.1 Genomes and chromosomes: An overview................................................................................1 1.2 B chromosomes..........................................................................................................................3 1.3 The enigma of B and sex chromosomes.....................................................................................5 1.4 Cichlids genomes: Interesting model systems for understanding B chromosomes....................8 1.5 Astatotilapia latifasciata as an exciting model species for B chromosomics............................9 1.6 The importance of chromosome-scale genome assembly for investigating B chromosome biology............................................................................................................................................10 1.7 3D genome organization and its importance in investigating the B chromosome biology......13 1.8 Rationale of the present study..................................................................................................17 2 OBJECTIVES AND HYPOTHESIS...............................................................................................19 2.1 General objectives....................................................................................................................19 2.2 Specific Objectives...................................................................................................................19 2.3 Hypothesis 1.............................................................................................................................20 2.4 Hypothesis 2.............................................................................................................................20 3 MATERIALS AND METHODS.....................................................................................................21 3.1 Ethics Statement.......................................................................................................................21 3.2 Fish samples and karyotype......................................................................................................21 3.3 PacBio circular consensus library preparation and sequencing................................................21 3.4 Hi-C library preparation...........................................................................................................22 3.5 Estimation of the genome size and sequencing coverage.........................................................23 3.6 The draft genome assembly of PacBio reads............................................................................23 3.7 Polishing draft assembly with Illumina reads..........................................................................24 3.8 Hi-C scaffolding and chromosome-scale assembly..................................................................25 3.9 Genome annotation...................................................................................................................27 3.9.1 Repeat element analysis and TEs identification................................................................27 3.9.2 Protein-coding gene structural annotation.........................................................................28 3.9.3 Functional annotation........................................................................................................28 3.10 B Chromosome identification.................................................................................................29 3.10.1 Genome alignments and coverage ratio analysis.............................................................29 3.10.2 Comparison of Hi-C interactions between 0B and 1B genomes....................................30 3.10.3 Mapping experimentally validated markers on B chromosome assembly......................30 3.11 3D genome organization and TADs detection........................................................................31 3.11.1 Comparison of 3D genome structure...............................................................................31 3.11.2 Preprocessing of Hi-C data and TAD prediction.............................................................31 3.11.3 A/B compartment analysis..............................................................................................33 3.11.4 Loop detection.................................................................................................................33 3.11.5 TADs and Loops size comparisons in both genomes......................................................34 3.11.6 Duplication breakpoints at the TADs...............................................................................34 3.11.7 Identification of TADs in representative cichlids species...............................................34 3.12 Comparative genomics analysis.............................................................................................35 3.12.1 Gene family clustering....................................................................................................35 3.12.2 Phylogeny and divergence time.......................................................................................35 3.12.3 Gene family evolution.....................................................................................................36 3.12.4 Genomic structural variation analysis.............................................................................37 3.13 Transcriptomic analysis..........................................................................................................37 3.13.1 B chromosome genes and differential expression..........................................................37 3.13.2 Analysis of genes transcription within and outside TADs...............................................38 3.14 Mapping of cichlid sex-determination genes in genome assembly........................................39 4 RESULTS........................................................................................................................................40 4.1 PacBio and Hi-C sequencing enabled a reference-quality chromosome-length assembly of A. latifasciata genome........................................................................................................................40 4.2 Genome assessment confirms the continuity and completeness of assembly..........................45 4.3 Chromosome localization of sex-determining genes suggests polygenic sex determination system in A. latifasciata.................................................................................................................48 4.4 Genome annotation and repeatomics revealed a low-level gene density and expansion of LTRs on B.......................................................................................................................................49 4.5 An integrative genomic approach allowed the isolation and validation of B chromosome sequences........................................................................................................................................52 4.6 Exploring the 3D genomics identified dynamics of chromatin organization in 1B genome. . .55 4.7 Comparative genomics provided adaptive evolutionary insights in cichlids...........................60 4.8 Genomic structural variation analysis revealed a higher rate of duplications in 1B as compared to 0B...............................................................................................................................67 4.9 B chromosome has lower genes expression than A chromosomes...........................................69 5 DISCUSSION..................................................................................................................................74 5.1 A chromosome scale cichlid genome for underpinning the B chromosome biology...............74 5.2 The hidden genomic content on B chromosome displays its evolutionary features................75 5.3 Does the B chromosome play any role in reshaping the chromatin conformation inside the cell?.................................................................................................................................................80 5.4 Cichlids genomes diversification is indicative of rapid adaptation and speciation..................84 5.5 The possibility of diverse range of sex determination systems in Lake Victoria cichlids........88 6 CONCLUSION...............................................................................................................................90 7 REFERENCES................................................................................................................................92 8 SUPPLEMENTARY DATA..........................................................................................................104 8.1 List of supplementary Figures................................................................................................104 8.2 List of supplementary Tables..................................................................................................105 LIST OF FIGURES Figure 1. Karyotype of A. latifasciata with B chromosomes..............................................................9 Figure 2. Hierarchical genome organization.....................................................................................14 Figure 3. HiC as a tool to study the effect of structural variation.....................................................16 Figure 4. An overview of assembly methods....................................................................................26 Figure 5. Chromosome scale assembly and the 3D model of A. latifasciata genome......................43 Figure 6. Evaluation of genome assemblies by multiple methods....................................................47 Figure 7. Circular visualization of genome assembly.......................................................................51 Figure 8. Validation and confirmation of B chromosome assembly.................................................54 Figure 9. The 3D genomic landscape of 0B and 1B genomes..........................................................57 Figure 10. Evolutionary trajectories of cichlid genomes...................................................................63 Figure 11. Genomic differences between the 0B and 1B genomes...................................................68 Figure 12. The transcriptional profiles of genes................................................................................73 LIST OF TABLES Table 1. Summary of obtained sequencing data……………………………………………………41 Table 2. A comparison of Illumina, PacBio and Hi-C de novo assemblies statistics……………….46 Table 3. Hi-C interactions of representative cichlids ………………………………………………60 Table 4. Functional enrichment of genes expansion on B chromosome…………………………...66 Table 5. Chromosome scale comparison of genes expression ……………………………………..71 ABSTRACT B chromosomes (Bs) are nonvital extra chromosomes found in diverse eukaryotic species including fungi, plants and animals. Among hundreds of investigated species, cichlid genomes offer fascinating models for studying B chromosome biology. Despite the extensive investigations, Bs are poorly understood mainly in relation to mechanisms of their own evolutionary survival as well as their structural and functional impact on genome organization. Our previous studies identified several sequences (genes and repeats) on the B of the cichlid fish Astatotilapia latifasciata but the complete genomic map of B chromosome has been missing. Here, we generated a chromosome- scale A. latifasciata genome with the B chromosome assembly by adopting integrative approach that combined deep coverage Pacific Biosciences single-molecule real-time (Pacbio long reads), high-throughput chromatin conformation capture (Hi-C) mapping, and Illumina (short-reads) sequencing. The assembled genome spans a total of 0.93 giga base pairs (Gb) genome with contig and scaffold N50 values of 3.4 and 36.2 mega base pairs (Mb) respectively. Compared with our previous Illumina based assembly, this upgraded genome is much more complete, and accurate. The annotation of core eukaryotic genes and universal single-copy orthologs has also been significantly improved and a total 150 Mb region has now been recovered, which was missing (in the previous assembly). We identified 759 protein-coding genes in the 34 Mb genomic content of B chromosome, of which at most of the genes showed a reduced level of expression as compared to A chromosomes. Our results demonstrate a substantial higher amount of transposable elements (TEs) mainly long terminal repeats (LTRs) retrotransposons (mean density 47.6) on the B chromosome as compared to the standard A chromosome set (mean density 10.45). We further applied whole- genome chromosome conformation capture (Hi-C) and in silico modeling methods to characterize the three dimensional (3D) architecture of and A and B chromosomes in the A. latifasciata genome. Remarkably, we observed a differential level organization of the chromatin into topologically associated domains (TADs) between 0B and 1B genomes. On a global level, the Hi-C interaction matrices of 1B genome are characterized by a relative gain of long-range and loss of short-range interactions within chromosomes indicating the impact of B chromosome on three-dimensional landscape of genome. Additionally, comparative analysis of A. latifasciata genome with other cichlids genomes enabled us to detect the phylogenomic diversification and chromosomal rearrangements including fusions and inversions providing interesting insights in co-ancestral cichlids gene evolution. Keywords: Cichlid genome; supernumerary chromosome; Hi-C; 3D genome, Genes evolution 6 CONCLUSION This study provides advances and contributions towards understanding the genomic composition structure, function and evolution of B chromosome in the cichlid fish. Generating a chromosome scale genome of A. latifasciata, our study provides crucial insights into B chromosome biology and reveals the genomic content of B chromosome updating significant additions to the sequences identified previously by Valente et al. (2014). The 3D genomic and bioinformatic approaches employed here broaden the study of karyotypes and chromosomes and fills the knowledge gaps in ways which were not possible using classical or even molecular cytogenetics alone. Long reads sequencing, and chromatin conformation capture technologies create new opportunities for understanding the function and evolution of sequences on B chromosomes. The genome assembly accomplished in the study significantly upgraded the previous version assembly recovering an addition of 150 Mb with a considerable 34 Mb of B chromosome content. The comprehensive genes annotations and repeat annotations using updated models and bioinformatic tools, described herein, will serve as a resource for expanding the compendium of B chromosome and reveal its impact on the host genome. The genes and repeatomic annotation found a high TEs density and low genes density on the B chromosome. Parallel to developing the annotation resource, we provide a methodological framework for assessing the three-dimensional genomic landscape of cichlid genome particularly in the context of the B chromosome dynamics inside the cell. The genomic and transcriptomics scale analyses together with exhaustive assessment of the Hi-C comparisons in the presence of B chromosome, sets the stage for functional studies to disentangle the role of B chromosome and for unlocking the mechanisms essential for driving the chromosome scale evolution. Deeper exploration of Hi-C data also suggests the complexity of genetic mechanisms in organization of TADs and further illustrate the myriad mechanisms by which B chromosome might become major contributor to induce differential TADs interactions in the host genome. Furthermore, 90 our transcriptomic profiling of B localized genes showed a reduced level expression, indicating the B chromosome may follow the partial inactivation, with exception of certain active genes which can represent advantages for B chromosome survival in the cell. Finally, the availability of the A. latifasciata chromosome scale genome assembly is also of great importance because it is very common fish in the aquarium, which is fortunate because it is now presumed extinct in the wild habitat of African lakes and also occupying a unique evolutionary position among cichlids species. In addition, the high-quality chromosome-level assembly obtained herein provides useful genetic information for accelerating the progress of B chromosome and sex chromosome origin, evolution and composition, as well as functional gene discovery and comparative genomics across the cichlid’s family. 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Genome size estimation generated by Genomescope, Figure S2. Hi-C interaction heatmap Figure S3. Comparison of A. latifascaiata chromosome level genome Figure S4. Insertion age of LTR elements in millions of years (Mya) Figure S5. Dot-plot comparison between the chr 2 and chr B Figure S6. Line plots show the chromosome-wise distribution of contact probability Figure S7. Chromosomes scale heatmap and bars plots of A/B compartments Figure S8. Hi-C heatmap based on the chromosome-scale assembly of the A. citrinellus (a) and O. niloticus Figure S9. Phylogenetic tree and estimated divergence times of cichlid species Figure S10. Graphical representation of summarized tree of the enriched gene ontology (GO) terms of CAFE expanded genes on B chromosome. Figure S11. Syntenic dotplots of A. citrinellus (left) and O. niloticus Figure S12. Comparative genomic sytenic analysis of M. zebra and A. aureus genomes Figure S13. The distribution of rearrangements Figure S14. Intra-chromosomal inversion Figure S15. Graph bar plot of normalized (TMM) expression level Figure S15. Comparison of DEGs between the gonads and muscles tissues 8.2 List of supplementary Tables Table S1. B_linked genes mapping Table S2. HiC-Explorer quality controls Table S3. cichlids_sex_determining_genes_list Table S4. GenomeScope results Table S5. Sample sequencing information 104 Table S6. Assemblies statistic comparison Table S7. Gene annotation and repeats contents Table S8. LAI score Table S9. Sex_linked genes mapping Table S10. Genome repeat landscape Table S11. LTRs evolutionary age Table S12. Cichlids chromsome level asemblies statistic Table S13. HiC based B-scaffolds Table S14. B_linked mapped genes hits on chrs Table S15. TADS_LOOPS_counts_cichlids Table S16. orthologous_genes_statistic Table S17. orthogroup_gene_count Table S18. A. latifascaita_expanded_genes_enrichment Table S19. A. latifascaita-contracted_genes_enrichment Table 20. B_expnded_genes_info Table S21. SV count per chr Table S22. FPKM_Pvalue_FDR_B-realted_genes Table S23. Brain_highly_sig_DEGs Table S24. Enriched_Bchr_genes 105 1 INTRODUCTION 1.1 Genomes and chromosomes: An overview 1.2 B chromosomes 1.3 The relationship between B and sex chromosomes 1.4 Cichlids genomes: Interesting model systems for understanding B chromosomes 1.5 Astatotilapia latifasciata as an exciting model species for B chromosomics 1.6 The importance of chromosome-scale genome assembly for investigating B chromosome biology 1.7 3D genome organization and its importance in investigating the B chromosome biology 1.8 Rationale of the present study 2 OBJECTIVES AND HYPOTHESIS 2.1 General objectives 2.2 Specific Objectives 2.3 Hypothesis 1 2.4 Hypothesis 2 3 MATERIALS AND METHODS 3.1 Ethics Statement 3.2 Fish samples and karyotype 3.3 PacBio circular consensus library preparation and sequencing 3.4 Hi-C library preparation 3.5 Estimation of the genome size and sequencing coverage 3.6 The draft genome assembly of PacBio reads 3.7 Polishing draft assembly with Illumina reads 3.8 Hi-C scaffolding and chromosome-scale assembly 1.1 Assembly Evaluation and Assessment 3.9 Genome annotation 3.9.1 Repeat element analysis and TEs identification 3.9.2 Protein-coding gene structural annotation 3.9.3 Functional annotation 3.10 B Chromosome identification 3.10.1 Genome alignments and coverage ratio analysis 3.10.2 Comparison of Hi-C interactions between 0B and 1B genomes 3.10.3 Mapping experimentally validated markers on B chromosome assembly 3.11 3D genome organization and TADs detection 3.11.1 Comparison of 3D genome structure 3.11.2 Preprocessing of Hi-C data and TAD prediction 3.11.3 A/B compartment analysis 3.11.4 Loop detection 3.11.5 TADs and Loops size comparisons in both genomes 3.11.6 Duplication breakpoints at the TADs 3.11.7 Identification of TADs in representative cichlids species 3.12 Comparative genomics analysis 3.12.1 Gene family clustering 3.12.2 Phylogeny and divergence time We estimated the divergence times between tree branches with penalized likelihood calculation implemented by the MCMCtree program in the PAML (Yang, 2000) package, using a Bayesian relaxed-molecular clock model. We checked for convergence and sufficient sampling using Tracer (Rambaut et al. 2018). 3.12.3 Gene family evolution 3.12.4 Genomic structural variation analysis Structural variation between the 0B and 1B genomes was identified through comparisons of assemblies of both genomes using SyRI v1.6 (Goel et al. 2019) software. The “nucmer” command was used for whole-genome alignment between 0B and 1B genomes. The parameter of “--maxmatch -c 500 -b 500 -l 50” was used in the command “nucmer” and the parameter of “-i 95 -l 1000 -m” in the command of delta-filter, which resulted in best alignments with at least 1 Kb matches and at least 95% identity between the two assembled genomes. The “show-coords” command with the parameter of “-THrd” was run to convert alignments to a tab-delimited flat text format. Alignment results were then used for identifying genomic structural variation and nucleotide polymorphisms through SyRI v1.6 (Goel et al. 2019) with the parameter of “--allow-offset 100”. Syri analysis discovered genome duplication, translocation, inversion, as well as syntenic, un- aligned, divergent sequences. SNPs, small insertions, and deletions were identified as well. 3.13 Transcriptomic analysis 3.13.1 B chromosome genes and differential expression We used the preprocessed (trimmed and filtered) RNA-seq data for 3 tissues (Go, gonad; Mu, Muscle; Br, Brain) of 0B and 1B individuals including both sexes of A. latifasciata. The transcriptomic data for these samples were originally generated by Nakajima, (2019). We aligned 0B and 1B RNA-seq Illumina reads to our chromosome level genome using STAR version=2.7.3a aligner (Dobin et al. 2013). Sam files were converted into BAM and then sorted (samtools sort) and indexed (samtools index) (Li et al. 2009). The alignment output BAM files were then processed for comparing the RNA expression levels in B chromosome genes versus A chromosomes and the reads mapped to B genes were enumerated using Bedtools “multicov” (Quinlan and Hall, 2010). We counted the number of mapped reads for all genes annotated on the B chromosome using Bedtools “multicov”. Expression values (log10 of mapped reads count) were calculated in R and the results were generated in a tab-delimited format. To test the significant difference among the expression level of chromosomes, we performed the Analysis of Variance (ANOVA) and compared the means of log10 of mapped reads count across all chromosome genes. We also assessed the differential expression of the B localized genes in the assembly for estimation of downregulated and upregulated genes in the transcriptomic (0B and 1B) data. RNA-Seq expression level was normalized and differential expression analysis was performed using EdgeR (version 3.36.0) (Robinson et al. 2010). The default parameters p-adj/FDR = 0.05; logFC = 2; CPM = 1 were used for normalization. These parameters were applied on each replica of the corresponding sample was carried out on all filtered differential expression genes (DEGs) by selecting a threshold of FDR-adjusted p-value less than 0.05. 3.13.2 Analysis of genes transcription within and outside TADs TAD boundaries at B genome were identified using HicExplorer, which calculated the boundaries according to an improved version of TAD-separation score method (Ramírez et al. 2018). More details of TAD boundaries separation method are reported in Ramirez et al. (2018). In order to analyze the transcription of genes at TAD boundaries, we used the same transcriptomic data (as described earlier). We tested whether overall the genes which lie within TADs may have altered expression level as compared to the genes localized outside the TADs. For this analysis, we extracted both the genes within the TADs and the genes which were located at 25 Kb upstream and downstream regions of the corresponding TADs boundaries. We used bedtools “intersectBed” to extract the RNA-seq alignments from the transcriptomic bam files and count mapped reads of genes within the selected regions using the same approach described above. Genes were considered expressed if they have a normalized log-count of 1 or more. 3.14 Mapping of cichlid sex-determination genes in genome assembly To identify the sex determining sequences, the nucleotide sequences of several SD (sex determination) genes previously identified on different chromosomes of cichlid fishes were retrieved from NCBI (Table S3). Sequences were aligned to the genome assembly of A. latifasciata using BLASTn (blast+ v2.6.0) (Camacho et al. 2009). First, a local database of A. latifasciata sequences was constructed using “make- blastdb -dbtype nucl.” Then, the sequences of SD genes were compared to this database and the best hit was retrieved using “blastn -outfmt 6 -evalue 1000.” The number of BLAST hits, E- values, and percent identity were evaluated for filtering out the random hits. All the mapped SD genes that passed the criteria of (percent identity > 80% and e-value < 0) were considered as “significant hit” and called as putative sex-linked orthologs. 4 RESULTS 4.1 PacBio and Hi-C sequencing enabled a reference-quality chromosome-length assembly of A. latifasciata genome 4.2 Genome assessment confirms the continuity and completeness of assembly 4.3 Chromosome localization of sex-determining genes suggests polygenic sex determination system in A. latifasciata 4.4 Genome annotation and repeatomics revealed a low-level gene density and expansion of LTRs on B 4.5 An integrative genomic approach allowed the isolation and validation of B chromosome sequences 4.6 Exploring the 3D genomics identified dynamics of chromatin organization in 1B genome 4.7 Comparative genomics provided adaptive evolutionary insights in cichlids 4.8 Genomic structural variation analysis revealed a higher rate of duplications in 1B as compared to 0B 4.9 B chromosome has lower genes expression than A chromosomes 5 DISCUSSION 5.1 A chromosome scale cichlid genome for underpinning the B chromosome biology 5.2 The hidden genomic content on B chromosome displays its evolutionary features 5.3 Does the B chromosome play any role in reshaping the chromatin conformation inside the cell? 5.4 Cichlids genomes diversification is indicative of rapid adaptation and speciation 5.5 The possibility of diverse range of sex determination systems in Lake Victoria cichlids 6 CONCLUSION 7 REFERENCES 8 SUPPLEMENTARY DATA 8.1 List of supplementary Figures 8.2 List of supplementary Tables