T-lex3 : An accurate tool to genotype and estimate population frequencies of transposable elements using the latest short-read whole genome sequencing data

Motivation: Transposable elements (TEs) constitute a significant proportion of the majority of genomes sequenced to date. TEs are responsible for a considerable fraction of the genetic variation within and among species. Accurate genotyping of TEs in genomes is therefore crucial for a complete identification of the genetic differences among individuals, populations and species. Results: In this work, we present a new version of T-lex, a computational pipeline that accurately genotypes and estimates the population frequencies of reference TE insertions using short-read high-throughput sequencing data. In this new version, we have re-designed the T-lex algorithm to integrate the BWA-MEM short-read aligner, which is one of the most accurate short-read mappers and can be launched on longer short-reads (e.g. reads >150 bp). We have added new filtering steps to increase the accuracy of the genotyping, and new parameters that allow the user to control both the minimum and maximum number of reads, and the minimum number of strains to genotype a TE insertion. We also showed for the first time that T-lex3 provides accurate TE calls in a plant genome. Availability and implementation: To test the accuracy of T-lex3, we called 1630 individual TE insertions in Drosophila melanogaster, 1600 individual TE insertions in humans, and 3067 individual TE insertions in the rice genome. We showed that this new version of T-lex is a broadly applicable and accurate tool for genotyping and estimating TE frequencies in organisms with different genome sizes and different TE contents. T-lex3 is available at Github: https://github.com/GonzalezLab/T-lex3

Mobile element insertions in rare diseases: a comparative benchmark and reanalysis of 60,000 exome samples

Mobile element insertions (MEIs) are a known cause of genetic disease but have been underexplored due to technical limitations of genetic testing methods. Various bioinformatic tools have been developed to identify MEIs in Next Generation Sequencing data. However, most tools have been developed specifically for genome sequencing (GS) data rather than exome sequencing (ES) data, which remains more widely used for routine diagnostic testing. In this study, we benchmarked six MEI detection tools (ERVcaller, MELT, Mobster, SCRAMble, TEMP2 and xTea) on ES data and on GS data from publicly available genomic samples (HG002, NA12878). For all the tools we evaluated sensitivity and precision of different filtering strategies. Results show that there were substantial differences in tool performance between ES and GS data. MELT performed best with ES data and its combination with SCRAMble increased substantially the detection rate of MEIs. By applying both tools to 10,890 ES samples from Solve-RD and 52,624 samples from Radboudumc we were able to diagnose 10 patients who had remained undiagnosed by conventional ES analysis until now. Our study shows that MELT and SCRAMble can be used reliably to identify clinically relevant MEIs in ES data. This may lead to an additional diagnosis for 1 in 3000 to 4000 patients in routine clinical ES

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution

A benchmark and an algorithm for detecting germline transposon insertions and measuring de novo transposon insertion frequencies

Transposons are genomic parasites, and their new insertions can cause instability and spur the evolution of their host genomes. Rapid accumulation of short-read whole-genome sequencing data provides a great opportunity for studying new transposon insertions and their impacts on the host genome. Although many algorithms are available for detecting transposon insertions, the task remains challenging and existing tools are not designed for identifying de novo insertions. Here, we present a new benchmark fly dataset based on PacBio long-read sequencing and a new method TEMP2 for detecting germline insertions and measuring de novo \u27singleton\u27 insertion frequencies in eukaryotic genomes. TEMP2 achieves high sensitivity and precision for detecting germline insertions when compared with existing tools using both simulated data in fly and experimental data in fly and human. Furthermore, TEMP2 can accurately assess the frequencies of de novo transposon insertions even with high levels of chimeric reads in simulated datasets; such chimeric reads often occur during the construction of short-read sequencing libraries. By applying TEMP2 to published data on hybrid dysgenic flies inflicted by de-repressed P-elements, we confirmed the continuous new insertions of P-elements in dysgenic offspring before they regain piRNAs for P-element repression. TEMP2 is freely available at Github: https://github.com/weng-lab/TEMP2

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’

IDENTIFYING AND CHARACTERIZING TRANSPOSABLE ELEMENTS IN THE GENOME

A large fraction of mammalian genome consists of transposable elements (TEs). These elements are segments of DNA that either move or are copied from one place in the genome to another. TEs are a significant source of genetic variation and are directly responsible for many diseases. It is difficult to identify, map, characterize, and determine the zygosity of TEs using current high-throughput short-read sequencing data because of their numerous copies in the genome. Existing approaches search for TE insertion (TEi) by aligning millions of mostly irrelevant short reads to either a reference genome or a TE sequence library. In this dissertation I describe two alignment-free novel TEi detection algorithms, ELITE and Frontier which outperform existing tools in several different categories. Both algorithms use local-genome-assembly where ELITE is template-dependent and Frontier is template-free. The key idea is to focus on identifying the boundary of TE insertion which contains partial TE and non-TE context. I use an msBWT-based data structure to store and index all the reads from a high-throughput sequencing dataset and leverages additional data structures FM-index and Longest Common Prefix (LCP) to efficiently search for TEi boundaries. I show that combination of two methods can identify nearly all the Endogenous RetoVirus (ERV) insertions that are segregating in a population with more than 100 samples. These methods can also be used to identify very recent or de novo TE insertions. Moreover, characterization based on the sharing pattern of ERVis allows us to study phylogeny within a population.Doctor of Philosoph

A Novel Approach to Epigenomic Profiling of Transposable Elements

ChIP-seq reads are notoriously difficult to assign to individual transposon copies. In particular, little is known about the locus-specific regulation of evolutionarily young transposable elements, which have been implicated in genome stability, gene regulation and innate immunity in a variety of developmental and disease contexts. Understanding of how individual copies are transcriptionally regulated cannot be appreciated without this information. To overcome this, I propose to leverage chromatin conformation information from Hi-ChIP data, a technique which combines Hi-C with ChIP-seq. As proximal (<50kb) chromatin interactions predominate, mappable genomic fragments are more likely to interact with repeats nearby than those lying far away. To implement this mapping strategy, we have developed PAtChER (Proximity-based alignment of Hi-ChIP ends to repeats). PAtChER employs information from Hi-ChIP chimeric reads to assign the multi-mapping read to a single location in the genome with high accuracy. Importantly, I demonstrate that PAtChER yields accurate protein enrichment profiles at individual repetitive loci. I then applied this to discover previously unappreciated protein positional information at individual transposable elements. This strategy will enable substantial improvements to our current understanding of transposon transcriptional regulation in health and disease and provide an invaluable tool to the transposon field