Bioinformatics and Next Generation Sequencing: Applications of Arthropod Genomes

Over the past decade, the Next Generation Sequencing (NGS) technology has been broadly applied in many areas such as genomics, medical diagnosis, biotechnology, virology, biological systematics, forensic biology, and anthropology. Taken together, it has offered us brilliant insights into life sciences. Most of the work presented in this thesis describes NGS applications on genome assembly, genome annotation, and comparative genomics, using arthropods as case studies: (1) by sequencing and analyzing the genomes of three Tetranychus spider mites with three completely different feeding behaviors, we uncovered genomic signature variations and indicative of pest adaptations; (2) we sequenced, assembled and annotated five Brevipalpus flat mite genomes and their corresponding endosymbiont Cardinium genomes. Comparative genomics reveals herbivorous pest adaptations and parthenogenesis; (3) the complete genomic analysis of parasitoid wasp Copidosoma floridanum indicates the mechanism of polyembryony of such primary parasite of moths. By bioinformatics and genomics approaches, my study provides the genomic basis and establishes the hypotheses for the future biology in pest and arthropod researches. These NGS applications of arthropod genomes will offer new insights into arthropod evolution and plant-herbivore interactions, open unique opportunities to develop novel plant protection strategies, and additionally, provide arthropod genomic resources as well

Advances in Single Molecule, Real-Time (SMRT) Sequencing

PacBio’s single-molecule real-time (SMRT) sequencing technology offers important advantages over the short-read DNA sequencing technologies that currently dominate the market. This includes exceptionally long read lengths (20 kb or more), unparalleled consensus accuracy, and the ability to sequence native, non-amplified DNA molecules. From fungi to insects to humans, long reads are now used to create highly accurate reference genomes by de novo assembly of genomic DNA and to obtain a comprehensive view of transcriptomes through the sequencing of full-length cDNAs. Besides reducing biases, sequencing native DNA also permits the direct measurement of DNA base modifications. Therefore, SMRT sequencing has become an attractive technology in many fields, such as agriculture, basic science, and medical research. The boundaries of SMRT sequencing are continuously being pushed by developments in bioinformatics and sample preparation. This book contains a collection of articles showcasing the latest developments and the breadth of applications enabled by SMRT sequencing technology

The Genome Sequence of Caenorhabditis briggsae: A Platform for Comparative Genomics

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes

Strukturell variasjon som påvirker genetisk miljøtilpasning i laksefisk

Structural variations (SVs), e.g. deletions, insertions, inversions and duplications of sequences, are a major source of genomic variation affecting more base pairs in the genome than single nucleotide polymorphisms (SNPs). Despite their increasingly recognised importance in adaptive evolution and species diversification, SVs are vastly understudied in most species. Long-read sequencing, together with recently developed bioinformatic tools, have provided step-change improvements in the precision and recall of SV detection and allow us to increase the detected SVs manyfold across the species range. In addition, long-reads represent a major shift in our ability to build continuous genome assemblies as fundamental resources for most genome wide studies. The work in this thesis utilises long-read data to generate multiple genome sequences for the two salmonid species Atlantic salmon (Salmo salar) and lake whitefish (Coregonus clupeaformis). We present the first pan-genome for Atlantic salmon, comprising 11 long-read-based assemblies across the species range. Among these, the highest quality genome has 2.55 Gbp assembled into chromosome sequences, 259 Mbp more sequence than in the previous Atlantic salmon reference genome. The genome has a highly improved continuity with contig N50 increasing from 58 kbp to 28.06 Mbp (484-fold). The detection of SVs in these 11 individuals, revealed 1,061,452 SVs, with an average of ~77.4 Mbp of sequence differing per sample. The Atlantic salmon has adapted to different river environment across a large geographical distribution. To investigate genomic variation underlying these adaptations, we associated SVs and environmental data in a dataset of 366 short-read samples genotyped using genome graph analyses. These analyses highlighted multiple SVs contributing to environmental adaptations, including an 18 kbp deletion encompassing a polymorphic segmental duplication of three genes associated with annual precipitation. Next, we use the Atlantic salmon pan-genome to study the emergence of supergenes. Because supergenes can be maintained over millions of years by balancing selection and typically exhibit strong recombination suppression, their underlying functional variants and how they are formed are largely unknown. Inversions are type of rearrangement commonly associated with supergenes, and by directly comparing multiple highly continuous genome assemblies we were able to detect a number of large inversions in Atlantic salmon. A 3 Mb inversion, estimated to be ~15,000-year-old, and segregating in North American populations, displayed supergene signatures with adaptive variation captured within the standard arrangement of the inversion, as well as other adaptive variation accumulating after the inversion occurred. Characterization of other inversions with matched repeat structures at the breakpoints did not show any supergene signatures, suggesting that shared breakpoint repeats may obstruct the supergene formation. Lastly, we created long-read based genome assemblies for sympatric species pairs (Dwarf and Normal) belonging to lake whitefish (Coregonus clupeaformis). The species pairs offer a suitable model system for studying genomic patterns of differentiation and in particular the role of SVs in speciation. By combining long-reads, direct assembly, and short-read methods we detect 89,909 high-confidence SVs in the species pair across two lakes, covering five times more sequence in the genome compared to SNPs. In the study, we highlight shared outliers of differentiation between the lakes, indicating that they contribute to speciation. Interestingly, we find that more than 70% of SVs differentiating between the Normal and Dwarf species pairs of lake whitefish are overlapping transposable elements. This work demonstrates that SVs may play an important role for the differentiation and speciation of sympatric species pairs in lake whitefish.Strukturell variasjon (SVer), for eksempel delesjoner, insersjoner, inversjoner og duplikasjoner av sekvens, er en viktig kilde til genomisk variasjon som samplet sett påvirker flere basepar i genomet enn punktmutasjoner (SNPs). Til tross for en økende annerkjennelse for at SVer spiller en viktig rolle i genetisk tilpassing til ulikt miljø og artsdannelse har denne typen variasjon vært lite studert i mange arter. Ny DNA-sekvenseringsteknologi med lengre leselengder (long-read sequencing), samt utvikling av nye bioinformatiske verktøy, har ført til drastiske forbedringer i deteksjonen av SVer. ‘Long-read’ sekvensering gjør det også mulig å lage mer komplette og sammenhengende genomsekvenser enn tidligere. I denne avhandlingen benytter vi oss av ‘long-read’ data til å lage flere genomsekvenser av høy kvalitet for to ulike laksefiskarter: Atlanterhavslaks (Salmo salar) og en Nordamerikansk type sik ‘lake whitefish’ (Coregonus clupeaformis). Her rapporterer vi det første pan-genomet for Atlanterhavslaks. Det består av 11 assemblier basert på ‘long- read’ sekvensering av individer fra fire ulike fylogeografiske grupper av villaks. Assembliet av høyest kvalitet inkluderer 2,55 Gbp sekvens i kromosomer, 259 Mbp mer enn det forrige referansegenomet til Atlanterhavslaks. I tillegg ble andelen sammenhengende sekvens, målt som contig N50, økt fra 58 kbp til 28,06 Mbp (484 ganger høyere). Vi fant 1.061.452 SVer på tvers av de 11 individene med ~77,4 Mbp gjennomsnittlig sekvensforskjell per prøve. Atlanterhavslaksen har over tid tilpasset miljøet i ulike elver. For å studere underliggende genetisk variasjon for denne tilpasningen assosierte vi SVer med ulike miljøvariabler i et datasett bestående av 366 ‘short-read’ sekvenserte prøver ved bruk av en genom-graf. Ved hjelp av disse analysene fant vi flere SVer som bidrar til miljøtilpasning, blant annet en 18 kbp lang delesjon som inneholder tre gener assosiert med mengden nedbør i området. Vi brukte så pan-genomet for Atlanterhavsaks til å studere dannelsen av ‘supergener’. Supergener er en sammenkobling av genetisk variasjon i koblingsulikevekt som for eksempel kan oppstå ved hjelp av store inversjoner. Her utnyttet vi 11 genomassemblier til å identifisere og karakterisere en rekke store inversjoner i Atlanterhavslaks. En av inversjonene på 3 Mbp, estimert til å være ~15.000 år gammel, viste signaturer for utvikling som supergen. For de andre inversjonene som var flankert av repetert DNA fant vi ikke karakteristiske trekk på supergener, noe som tyder på at det repetitive DNA forhindrer en dannelse av supergener. Til slutt lagde vi genomsekvenser for ulike former (‘Normal’ og ‘Dwarf’) av ‘lake whitefish’ (Coregonus clupeaformis) som lever i de samme innsjøene i Nord-Amerika. Genomsekvensene muliggjør studier av genomiske mekanismene bak artsdannelse i denne laksefisken. Ved å kombinere ‘long-read’ data, direkte sammenlikning av assemblier, og ‘short-read’ data fant vi 89,909 SVer som skilte de to formene av ‘lake whitefish’ i to innsjøer. SVene omfatter mer enn fem ganger flere basepar i genomet sammenlignet med SNPs. I studiet fant vi flere SVer med avvikende forekomst (‘outliers’) i de to formene av ‘lake whitefish’, noe som indikerer at disse SVene bidrar til artsdannelse. Videre fant vi at 70 % av SVene overlappet en form av repetert DNA kalt transposable elementer. Dette arbeidet understreker at SVer kan spille en viktig rolle for artsdannelse i ’lake whitefish’

Rapid Evolution of Enormous, Multichromosomal Genomes in Flowering Plant Mitochondria with Exceptionally High Mutation Rates

A pair of species within the genus Silene have evolved the largest known mitochondrial genomes, coinciding with extreme changes in mutation rate, recombination activity, and genome structure

Trypanosoma cruzi genome plasticity and evolution

Trypanosoma cruzi, a protozoan from the Kinetoplastidae family, is the etiologic agent of Chagas disease, a major public health problem affecting mostly the poorest areas of Latin America. Due to the complex nature of the parasite’s genome it has been impossible to produce a complete reference genome sequence, thus hampering the implementation of post- genomic approaches to unveil the mechanisms of generation of antigenic variation and the identification of new drug targets. My doctoral studies have focused on the application of combined genome sequencing and computational methods to produce a complete reference T. cruzi genome sequence and perform comparative analyses to better understand the mechanisms that allow T. cruzi to evade the mammalian host immune system and to briskly adapt to novel environments. In paper I and II, different genome assembly strategies and second generation sequencing technologies were implemented to perform comparative analyses to identify elements of virulence between T. cruzi and two trypanosomatids that are non-pathogenic to humans: Trypanosoma cruzi marinkellei, a bat-restricted sub-species of the T. cruzi clade and the human avirulent species Trypanosoma rangeli. The studies reveal the expansion of T. cruzi- specific genomic traits specialised in the invasion of mammalian cells. In paper III, using third-generation, PacBio sequencing data it was possible to assemble the complete reference genome sequence of a Trypanosoma cruzi isolate from the DTU-I clade. This breakthrough allowed us - for the first time - to explore in detail the genome architecture of the subtelomeric areas where many parasite virulence factors are encoded. One of the most interesting discoveries was the overrepresentation of interspersed retrotransposons and microsatellites in tandem gene arrays coding for surface molecules, hinting at a retrotransposon-driven mechanism of recombination for generating new sequence variants. Whole genome sequencing of 35 T. cruzi DTU-I isolates, collected from different locations in the American continent, made possible to identify and characterise the mechanisms of adaptability employed by the parasite. Finally, paper IV analyses the mechanisms of genomic hybridisation in T. cruzi and the evolution over time of the hybrid offspring. The analysis revealed that during hybrid formation, the parasite integrates genetic material from each parental strains with the aid of retrotransposons and microsatellites, and the genome of these hybrid isolates moves quickly from a tetraploid to a diploid state. As a result, the hybrid strain has more genetic material, mostly in the subtelomeres, providing the parasite with a pool of new surface molecule genes with the potential to possibly increase its fitness in a new environment. In conclusion, the work presented here has advanced the understanding of parasite biology and provided a genomic resource to be exploited for the identification of drug targets and vaccine candidates

Computational analysis of human genomic variants and lncRNAs from sequence data

The high-throughput sequencing technologies have been developed and applied to the human genome studies for nearly 20 years. These technologies have provided numerous research applications and have significantly expanded our knowledge about the human genome. In this thesis, computational methods that utilize sequence data to study human genomic variants and transcripts were evaluated and developed. Indel represents insertion and deletion, which are two types of common genomic variants that are widespread in the human genome. Detecting indels from human genomes is the crucial step for diagnosing indel related genomic disorders and may potentially identify novel indel makers for studying certain diseases. Compared with previous techniques, the high-throughput sequencing technologies, especially the next- generation sequencing (NGS) technology, enable to detect indels accurately and efficiently in wide ranges of genome. In the first part of the thesis, tools with indel calling abilities are evaluated with an assortment of indels and different NGS settings. The results show that the selection of tools and NGS settings impact on indel detection significantly, which provide suggestions for tool selection and future developments. In bioinformatics analysis, an indel’s position can be marked inconsistently on the reference genome, which may result in an indel having different but equivalent representations and cause troubles for downstream. This problem is related to the complex sequence context of the indels, for example, short tandem repeats (STRs), where the same short stretch of nucleotides is amplified. In the second part of the thesis, a novel computational tool VarSCAT was described, which has various functions for annotating the sequence context of variants, including ambiguous positions, STRs, and other sequence context features. Analysis of several high- confidence human variant sets with VarSCAT reveals that a large number of genomic variants, especially indels, have sequence features associated with STRs. In the human genome, not all genes and their transcripts are translated into proteins. Long non-coding ribonucleic acid (lncRNA) is a typical example. Sequence recognition built with machine learning models have improved significantly in recent years. In the last part of the thesis, several machine learning-based lncRNA prediction tools were evaluated on their predictions for coding potentiality of transcripts. The results suggest that tools based on deep learning identify lncRNAs best. Ihmisen genomivarianttien ja lncRNA:iden laskennallinen analyysi sekvenssiaineistosta Korkean suorituskyvyn sekvensointiteknologioita on kehitetty ja sovellettu ihmisen genomitutkimuksiin lähes 20 vuoden ajan. Nämä teknologiat ovat mahdollistaneet ihmisen genomin laaja-alaisen tutkimisen ja lisänneet merkittävästi tietoamme siitä. Tässä väitöstyössä arvioitiin ja kehitettiin sekvenssiaineistoa hyödyntäviä laskennallisia menetelmiä ihmisen genomivarianttien sekä transkriptien tutkimiseen. Indeli on yhteisnimitys lisäys- eli insertio-varianteille ja häviämä- eli deleetio-varianteille, joita esiintyy koko genomin alueella. Indelien tunnistaminen on ratkaisevaa geneettisten poikkeavuuksien diagnosoinnissa ja eri sairauksiin liittyvien uusien indeli-markkereiden löytämisessä. Aiempiin teknologioihin verrattuna korkean suorituskyvyn sekvensointiteknologiat, erityisesti seuraavan sukupolven sekvensointi (NGS) mahdollistavat indelien havaitsemisen tarkemmin ja tehokkaammin laajemmilta genomialueilta. Väitöstyön ensimmäisessä osassa indelien kutsumiseen tarkoitettuja laskentatyökaluja arvioitiin käyttäen laajaa valikoimaa indeleitä ja erilaisia NGS-asetuksia. Tulokset osoittivat, että työkalujen valinta ja NGS-asetukset vaikuttivat indelien tunnistukseen merkittävästi ja siten ne voivat ohjata työkalujen valinnassa ja kehitystyössä. Bioinformatiivisessa analyysissä saman indelin sijainti voidaan merkitä eri kohtiin referenssigenomia, joka voi aiheuttaa ongelmia loppupään analyysiin, kuten indeli-kutsujen arviointiin. Tämä ongelma liittyy sekvenssikontekstiin, koska variantit voivat sijoittua lyhyille perättäisille tandem-toistojaksoille (STR), jossa sama lyhyt nukleotidijakso on monistunut. Väitöstyön toisessa osassa kehitettiin laskentatyökalu VarSCAT, jossa on eri toimintoja, mm. monitulkintaisten sijaintitietojen, vierekkäisten alueiden ja STR-alueiden tarkasteluun. Luotettaviksi arvioitujen ihmisen varianttiaineistojen analyysi VarSCAT-työkalulla paljasti, että monien geneettisten varianttien ja erityisesti indelien ominaisuudet liittyvät STR-alueisiin. Kaikkia ihmisen geenejä ja niiden geenituotteita, kuten esimerkiksi ei-koodaavia RNA:ta (lncRNA) ei käännetä proteiiniksi. Koneoppimismenetelmissä ja sekvenssitunnistuksessa on tapahtunut huomattavaa parannusta viime vuosina. Väitöstyön viimeisessä osassa arvioitiin useiden koneoppimiseen perustuvien lncRNA-ennustustyökalujen ennusteita. Tulokset viittaavat siihen, että syväoppimiseen perustuvat työkalut tunnistavat lncRNA:t parhaiten

Repeat expansions in leukoencephalopathy

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152577/1/ana25613.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152577/2/ana25613_am.pd