Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the C¸ ukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle

The genome of Leishmania adleri from a mammalian host highlights chromosome fission in Sauroleishmania

Control of pathogens arising from humans, livestock and wild animals can be enhanced by genome-based investigation. Phylogenetically classifying and optimal construction of these genomes using short sequence reads are key to this process. We examined the mammal-infecting unicellular parasite Leishmania adleri belonging to the lizard-infecting Sauroleishmania subgenus. L. adleri has been associated with cutaneous disease in humans, but can be asymptomatic in wild animals. We sequenced, assembled and investigated the L. adleri genome isolated from an asymptomatic Ethiopian rodent (MARV/ET/75/HO174) and verified it as L. adleri by comparison with other Sauroleishmania species. Chromosome- level scaffolding was achieved by combining reference-guided with de novo assembly followed by extensive improvement steps to produce a final draft genome with contiguity comparable with other references. L. tarentolae and L. major genome annotation was transferred and these gene models were manually verified and improved. This first high-quality draft Leishmania adleri reference genome is also the first Sauroleishmania genome from a non-reptilian host. Comparison of the L. adleri HO174 genome with those of L. tarentolae Parrot-TarII and lizard-infecting L. adleri RLAT/KE/1957/SKINK-7 showed extensive gene amplifications, pervasive aneuploidy, and fission of chromosomes 30 and 36. There was little genetic differentiation between L. adleri extracted from mammals and reptiles, highlighting challenges for leishmaniasis surveillance

Inherent Mosaicism and Extensive Mutation of Human Placentas

Approximately 1% to 2% of pregnancies involve chromosomal aberrations confined to the placenta. During the first few days of embryogenesis when fetal and placental lineages diverge, clonal expansion and mosaicism may occur, representing a potential pathological divergence from normal clonal dynamics of early embryogenesis. Mutations acquired during cell division may be identified and used to reconstruct embryonic lineage relationships that can reveal specific mutagenic processes affecting a tissue

Suunatud ja ülegenoomsel sekveneerimisel põhinevate mitteinvasiivsete sünnieelsete testide arvutusmeetodite ja töövoogude väljatöötamine

Väitekirja elektrooniline versioon ei sisalda publikatsiooneLoote sõeluuring võimaldab avastada lootel esinevaid arenguhäireid ja sagedasemaid kromosoomhaiguseid, nagu näiteks Down’i, Edwards’i ja Patau sündroom. Varajane teave lootel esineva kromosoomhaiguse kohta võimaldab langetada informeeritud otsust raseduse jätkamise osas ning aitab tulevasi vanemaid paremini ette valmistada. Tavapärane loote sõeluuring sisaldab loote ultraheli uuringut ja vereseerumi analüüsi, mille abil tuvastatakse enamik kromosoomhaigusega loodetest. Lõpliku diagnoosi saamiseks suunatakse kõrge riski saanud patsient edasi invasiivsele protseduurile. Eelnimetatud sõeluuringute puuduseks on arvestatav valepositiivsete hulk, mistõttu enamik positiivse testitulemuse saanud patsientidest kannab täiesti tervet loodet. Sõeluuringule järgnev invasiivne protseduur on neil juhtudel ebavajalik, põhjustab rasedatele asjatut stressi ning sellega võib kaasneda suurenenud oht raseduse katkemiseks. Antud doktoritöö keskseks teemaks on mitte-invasiivne sünnieelne testimine (NIPT), mis põhineb ema veres leiduva loote päritolu rakuvaba DNA analüüsil. Võrreldes eelmainitud traditsionaalsete sõeluuringu meetoditega, on NIPT oluliselt sensitiivsem ja spetsiifilisem sagedamini esinevate kromosoomihäirete avastamiseks. Doktoritöö raames arendati välja TAC-seq põhine analüüsi töövoog, mida rakendati 21. kromosoom trisoomia tuvastamiseks. Lisaks töötati välja NIPT analüüsiraamistik, mis kasutab erinevaid masinõppe metoodikaid loote trisoomia määramiseks rakuvaba DNA-st. Niisamuti viidi Eesti rasedate kohordil läbi NIPT metoodika validatsiooni uuring, milles rakendati ülegenoomsel sekveneerimisel põhinevat töövoogu sagedamate loote kromosoomihäirete määramiseks. Üldiselt on nii suunatud kui ka ülegenoomsel NIPT meetoditel muutnud rasedate sõeluuring varasemast veel täpsemaks. Kui suunatud sekveneerimise suureks eeliseks on kulutõhusus, siis ülegenoomne lähenemine tuvastab valimatult kõikvõimalikke geneetilisi aberratsioone üle kogu genoomi.Fetal screening allows to detect congenital anomalies and more frequent chromosomal abnormalities, such as Down, Edwards and Patau syndrome. Early information about a fetus’s possible health problem allows to make an informed decision about the continuation of the pregnancy and better prepare the future parents. Conventional screening includes an ultrasound and blood serum analysis by way of which most of the fetal chromosomal abnormalities are detected. For a final diagnosis, the patients who are deemed to have a high risk for fetal chromosomal aberrations are referred to an invasive procedure. The disadvantage of the aforementioned screening method is a considerable number of false positive results, which is why most of the patients who receive a positive result are actually carrying a fully healthy fetus. The invasive procedure that follows the screening is unnecessary for those patients, causes them undue stress and this may also lead to a higher risk of miscarriage. The focal point of this doctoral thesis is non-invasive prenatal testing (NIPT), which is based on the analysis of cell-free DNA (cfDNA) of fetal origin that is found in maternal blood. In comparison to the above-mentioned conventional screening methods, NIPT is considerably more sensitive and specific for detecting the most common chromosomal abnormalities. In the framework of the thesis, TAC-seq based analysis workflow was developed and used to detect chromosome 21 trisomy. In addition, NIPT analysis framework, which uses different machine learning methods, was developed for determining fetal trisomies from cfDNA sample. Also, a validation study of NIPT was carried out on pregnant women in Estonian cohort using a whole-genome sequencing based workflow. In general, both targeted and whole-genome sequencing based NIPT methods have made prenatal screening of fetal aneuplodies even more accurate than before. While cost-effectiveness is a major advantage of the targeted sequencing based approach, the whole-genome sequencing based NIPT possibly detects all kinds of genetic aberrations across the genome.https://www.ester.ee/record=b549777

Single-cell sequencing of genomic DNA resolves sub-clonal heterogeneity in a melanoma cell line

Abstract: We performed shallow single-cell sequencing of genomic DNA across 1475 cells from a cell-line, COLO829, to resolve overall complexity and clonality. This melanoma tumor-line has been previously characterized by multiple technologies and is a benchmark for evaluating somatic alterations. In some of these studies, COLO829 has shown conflicting and/or indeterminate copy number and, thus, single-cell sequencing provides a tool for gaining insight. Following shallow single-cell sequencing, we first identified at least four major sub-clones by discriminant analysis of principal components of single-cell copy number data. Based on clustering, break-point and loss of heterozygosity analysis of aggregated data from sub-clones, we identified distinct hallmark events that were validated within bulk sequencing and spectral karyotyping. In summary, COLO829 exhibits a classical Dutrillaux’s monosomic/trisomic pattern of karyotype evolution with endoreduplication, where consistent sub-clones emerge from the loss/gain of abnormal chromosomes. Overall, our results demonstrate how shallow copy number profiling can uncover hidden biological insights

A Study of the Bovine 1/29 Robertsonian Translocation Chromosome

Three hundred microscope slides containing fixed chromosome spreads from cattle with the normal karyotype were treated by either of two methods used to produce G-bands. Unsatisfactory results were obtained using one of these methods but distinct bands suitable for the identification of individual chromosomes were produced using the other. Karyotypes were prepared from 25 of the most clearly stained chromosome sets and the banding pattern of each individual chromosome was described in detail. Karyotypes were also prepared from 20 sets of C-banded chromosomes

Lineage tracing of normal human development and childhood cancers

From fertilisation onwards, the cells of the human body continuously experience damage to their genome, either from intrinsic causes or from exposure to mutagens. While the vast majority of DNA damage is repaired and the genome is replicated with extremely high fidelity, cells steadily acquire single nucleotide variants throughout life. Since cells pass these genetic changes on to their descendants, mutations shared between any two cells therefore imply a shared developmental path. In essence, these somatic mutations connect all cells together into one large phylogenetic tree of human development with the zygote at the root. Reconstructing phylogenies of human development requires readouts of somatic mutations present in single cells. Recently, low-input whole-genome sequencing following laser-capture microdissection has allowed us to reliably call somatic mutations in distinct single-cell derived physiological units, such as colonic crypts and endometrial glands, while retaining spatial information on a microscopic level. In this way, I reconstructed large-scale phylogenies of cells from many different organs of three individuals. These phylogenetic trees recapitulate the early stages of embryonic development and asymmetric cell allocation in the blastocyst, as well as later clonal expansions such as benign prostatic hyperplasia and neoplastic polyp formation. In a similar way, I also used somatic mutations to investigate the emergence of paediatric cancer, which is thought to be closely linked to aberrations in development. In the context of phylogenetic analyses of tumours, mutations shared between childhood cancers and different normal tissues can shed light on the embryonic lineage of tumours and may reveal the precise juncture at which tumours began to form. Accordingly, I studied the origin of Wilms tumour, the most common childhood cancer of the kidney. I discovered that these tumours often arise from large tissue-resident precursor clones residing in the normal kidney. These embryonal precursors represent an early clonal expansion driven by H19 hypermethylation. Lastly, using somatic mutations I discovered that the human placenta is made up of large clonal patches of closely related trophoblast cells. Comparing early embryonic mutations between placental lineages and umbilical cord DNA, which is derived from the inner cell mass, revealed that in approximately half of the cases, a trophectodermal lineage shares no somatic mutations with the umbilical cord. Furthermore, in a quarter of cases, the umbilical cord is entirely derived from a progenitor later than the zygote. This indicates a natural early segregation between these lineages and a pathway to generate confined placental mosaicism. This dissertation as a whole provides a new framework to study normal and aberrant human development from whole-genome sequencing. The ability to reconstruct developmental lineages retrospectively can answer fundamental questions about human development and carcinogenesis

Evaluation of the possibility to detect fetal chromosome trisomies based on a defined set of single nucleotide polymorphisms for non-invasive prenatal testing

Non-invasive prenatal testing (NIPT) of fetal aneuploidy using cell-free fetal (cffDNA) from mother’s blood sample has shown to be an accurate and reliable screening tool. The current NIPT protocols are based on targeted or whole genome sequencing, which demand resource-intensive bioinformatical capacity. The complexity of current NIPT technology is trustworthy but the comprehensive adaption of the application is still limited due to the high cost. Mother- and fetus-specific genotypes, according to the nature of cell-free DNA (cfDNA) during pregnancy, were simulated and used in further analysis. Simulations and theoretical calculations demonstrate the characteristic patterns of allelic ratios in case of normal number of chromosomes or trisomy where extra chromosome is inherited from mother or father. Here described analytical approach managed to identify fetal trisomy by comparing the allelic ratios of the risk chromosome with the expected allelic ratios using the t-test and hidden Markov model (HMM) analysis. An average, 3/4 of all highly polymorphic single nucleotide polymorphisms (SNPs) can be used in analysis based on comparison of the allelic ratios. As a result, at least 300 highly polymorphic SNPs over risk and reference chromosomes are needed to detect fetal trisomy using t-test alone. In addition, the HMM analysis can independently detect fetal trisomy and have the ability to distinguish the parental origin of trisomy. Based on the simulated data, the existence and the origin of fetal trisomy is theoretically detectable using a novel and highly quantitative SNP-based approach that is under development by our research group. However, further testing has to be carried out with the real data to confirm the theory