The Application of Long-Read Sequencing to Cancer.

Cancer is a multifaceted disease arising from numerous genomic aberrations that have been identified as a result of advancements in sequencing technologies. While next-generation sequencing (NGS), which uses short reads, has transformed cancer research and diagnostics, it is limited by read length. Third-generation sequencing (TGS), led by the Pacific Biosciences and Oxford Nanopore Technologies platforms, employs long-read sequences, which have marked a paradigm shift in cancer research. Cancer genomes often harbour complex events, and TGS, with its ability to span large genomic regions, has facilitated their characterisation, providing a better understanding of how complex rearrangements affect cancer initiation and progression. TGS has also characterised the entire transcriptome of various cancers, revealing cancer-associated isoforms that could serve as biomarkers or therapeutic targets. Furthermore, TGS has advanced cancer research by improving genome assemblies, detecting complex variants, and providing a more complete picture of transcriptomes and epigenomes. This review focuses on TGS and its growing role in cancer research. We investigate its advantages and limitations, providing a rigorous scientific analysis of its use in detecting previously hidden aberrations missed by NGS. This promising technology holds immense potential for both research and clinical applications, with far-reaching implications for cancer diagnosis and treatment.

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts.

BACKGROUND: A major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials. METHODS: Here, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry, and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors. RESULTS: We show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood-brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components. CONCLUSIONS: Our data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment.

Plasticity of repetitive sequences demonstrated by the complete mitochondrial genome of Eucalyptus camaldulensis.

The tree Eucalyptus camaldulensis is a ubiquitous member of the Eucalyptus genus, which includes several hundred species. Despite the extensive sequencing and assembly of nuclear genomes from various eucalypts, the genus has only one fully annotated and complete mitochondrial genome (mitogenome). Plant mitochondria are characterized by dynamic genomic rearrangements, facilitated by repeat content, a feature that has hindered the assembly of plant mitogenomes. This complexity is evident in the paucity of available mitogenomes. This study, to the best of our knowledge, presents the first E. camaldulensis mitogenome. Our findings suggest the presence of multiple isomeric forms of the E. camaldulensis mitogenome and provide novel insights into minor rearrangements triggered by nested repeat sequences. A comparative sequence analysis of the E. camaldulensis and E. grandis mitogenomes unveils evolutionary changes between the two genomes. A significant divergence is the evolution of a large repeat sequence, which may have contributed to the differences observed between the two genomes. The largest repeat sequences in the E. camaldulensis mitogenome align well with significant yet unexplained structural variations in the E. grandis mitogenome, highlighting the adaptability of repeat sequences in plant mitogenomes.

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts.

Background: A major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials. Methods: Here, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA-sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors. Results: We show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood-brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components. Conclusions: Our data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment.

Evolutionary selection of alleles in the melanophilin gene that impacts on prostate organ function and cancer risk.

BACKGROUND AND OBJECTIVES: Several hundred inherited genetic variants or SNPs that alter the risk of cancer have been identified through genome-wide association studies. In populations of European ancestry, these variants are mostly present at relatively high frequencies. To gain insight into evolutionary origins, we screened a series of genes and SNPs linked to breast or prostate cancer for signatures of historical positive selection. METHODOLOGY: We took advantage of the availability of the 1000 genome data and we performed genomic scans for positive selection in five different Caucasian populations as well as one African reference population. We then used prostate organoid cultures to provide a possible functional explanation for the interplay between the action of evolutionary forces and the disease risk association. RESULTS: Variants in only one gene showed genomic signatures of positive, evolutionary selection within Caucasian populations melanophilin (MLPH). Functional depletion of MLPH in prostate organoids, by CRISPR/Cas9 mutation, impacted lineage commitment of progenitor cells promoting luminal versus basal cell differentiation and on resistance to androgen deprivation. CONCLUSIONS AND IMPLICATIONS: The MLPH variants influencing prostate cancer risk may have been historically selected for their adaptive benefit on skin pigmentation but MLPH is highly expressed in the prostate and the derivative, positively selected, alleles decrease the risk of prostate cancer. Our study suggests a potential functional mechanism via which MLPH and its genetic variants could influence risk of prostate cancer, as a serendipitous consequence of prior evolutionary benefits to another tissue. LAY SUMMARY: We screened a limited series of genomic variants associated with breast and prostate cancer risk for signatures of historical positive selection. Variants within the melanophilin (MLPH) gene fell into this category. Depletion of MLPH in prostate organoid cultures, suggested a potential functional mechanism for impacting on cancer risk, as a serendipitous consequence of prior evolutionary benefits to another tissue.

LeafGo: Leaf to Genome, a quick workflow to produce high-quality de novo plant genomes using long-read sequencing technology.

Currently, different sequencing platforms are used to generate plant genomes and no workflow has been properly developed to optimize time, cost, and assembly quality. We present LeafGo, a complete de novo plant genome workflow, that starts from tissue and produces genomes with modest laboratory and bioinformatic resources in approximately 7 days and using one long-read sequencing technology. LeafGo is optimized with ten different plant species, three of which are used to generate high-quality chromosome-level assemblies without any scaffolding technologies. Finally, we report the diploid genomes of Eucalyptus rudis and E. camaldulensis and the allotetraploid genome of Arachis hypogaea.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

LongQC: A Quality Control Tool for Third Generation Sequencing Long Read Data.

We propose LongQC as an easy and automated quality control tool for genomic datasets generated by third generation sequencing (TGS) technologies such as Oxford Nanopore technologies (ONT) and SMRT sequencing from Pacific Bioscience (PacBio). Key statistics were optimized for long read data, and LongQC covers all major TGS platforms. LongQC processes and visualizes those statistics automatically and quickly.

Single cell analysis of clonal architecture in acute myeloid leukaemia.

We used single cell Q-PCR on a micro-fluidic platform (Fluidigm) to analyse clonal, genetic architecture and phylogeny in acute myeloid leukaemia (AML) using selected mutations. Ten cases of NPM1c mutant AML were screened for 111 mutations that are recurrent in AML and cancer. Clonal architectures were relatively simple with one to six sub-clones and were branching in some, but not all, patients. NPM1 mutations were secondary or sub-clonal to other driver mutations (DNM3TA, TET2, WT1 and IDH2) in all cases. In three of the ten cases, single cell analysis of enriched CD34+/CD33- cells revealed a putative pre-leukaemic sub-clone, undetectable in the bulk CD33+ population that had one or more driver mutations but lacked NPM1c. Cells from all cases were transplanted into NSG mice and in most (8/10), more than one sub-clone (#2-5 sub-clones) transplanted. However, the dominant regenerating sub-clone in 9/10 cases was NPM1+ and this sub-clone was either dominant or minor in the diagnostic sample from which it was derived. This study provides further evidence, at the single cell level, for genetic variegation in sub-clones and stem cells in acute leukaemia and demonstrates both a preferential order of mutation accrual and parallel evolution of sub-clones.

The Spatiotemporal Evolution of Lymph Node Spread in Early Breast Cancer.

Purpose: The most significant prognostic factor in early breast cancer is lymph node involvement. This stage between localized and systemic disease is key to understanding breast cancer progression; however, our knowledge of the evolution of lymph node malignant invasion remains limited, as most currently available data are derived from primary tumors.Experimental Design: In 11 patients with treatment-naïve node-positive early breast cancer without clinical evidence of distant metastasis, we investigated lymph node evolution using spatial multiregion sequencing (n = 78 samples) of primary and lymph node deposits and genomic profiling of matched longitudinal circulating tumor DNA (ctDNA).Results: Linear evolution from primary to lymph node was rare (1/11), whereas the majority of cases displayed either early divergence between primary and nodes (4/11) or no detectable divergence (6/11), where both primary and nodal cells belonged to a single recent expansion of a metastatic clone. Divergence of metastatic subclones was driven in part by APOBEC. Longitudinal ctDNA samples from 2 of 7 subjects with evaluable plasma taken perioperatively reflected the two major evolutionary patterns and demonstrate that private mutations can be detected even from early metastatic nodal deposits. Moreover, node removal resulted in disappearance of private lymph node mutations in ctDNA.Conclusions: This study sheds new light on a crucial evolutionary step in the natural history of breast cancer, demonstrating early establishment of axillary lymph node metastasis in a substantial proportion of patients. Clin Cancer Res; 24(19); 4763-70. ©2018 AACR.

The subclonal complexity of STIL-TAL1+ T-cell acute lymphoblastic leukaemia.

Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.

Less effective selection leads to larger genomes.

The evolutionary origin of the striking genome size variations found in eukaryotes remains enigmatic. The effective size of populations, by controlling selection efficacy, is expected to be a key parameter underlying genome size evolution. However, this hypothesis has proved difficult to investigate using empirical data sets. Here, we tested this hypothesis using 22 de novo transcriptomes and low-coverage genomes of asellid isopods, which represent 11 independent habitat shifts from surface water to resource-poor groundwater. We show that these habitat shifts are associated with higher transcriptome-wide [Formula: see text] After ruling out the role of positive selection and pseudogenization, we show that these transcriptome-wide [Formula: see text] increases are the consequence of a reduction in selection efficacy imposed by the smaller effective population size of subterranean species. This reduction is paralleled by an important increase in genome size (25% increase on average), an increase also confirmed in subterranean decapods and mollusks. We also control for an adaptive impact of genome size on life history traits but find no correlation between body size, or growth rate, and genome size. We show instead that the independent increases in genome size measured in subterranean isopods are the direct consequence of increasing invasion rates by repeat elements, which are less efficiently purged out by purifying selection. Contrary to selection efficacy, polymorphism is not correlated to genome size. We propose that recent demographic fluctuations and the difficulty of observing polymorphism variation in polymorphism-poor species can obfuscate the link between effective population size and genome size when polymorphism data are used alone.

Systematic assessment of the influence of complement gene polymorphisms on kidney transplant outcome.

The importance of the innate immune system, including complement, in causing transplant injury and augmenting adaptive immune responses is increasingly recognized. Therefore variability in graft outcome may in part be due to genetic polymorphism in genes encoding proteins of the immune system. This study assessed the relationship between single nucleotide polymorphisms (SNPs) in complement genes and outcome after transplantation. Analysis was performed on two patient cohorts of 650 and 520 transplant recipients. 505 tagged SNPs in 47 genes were typed in both donor and recipient. The relationships between SNPs and graft survival, serum creatinine, delayed graft function and acute rejection were analyzed. One recipient SNP in the gene encoding mannose binding lectin was associated with graft outcome after correction for analysis of multiple SNPs (p=6.41 × 10(-5)). When further correction was applied to account for analysis of the effect of SNPs in both donor and recipient this lost significance. Despite association p values of <0.001 no SNP was significantly associated with clinical phenotypes after Bonferroni correction. In conclusion, the variability seen in transplant outcome in this patient cohort cannot be explained by variation in complement genes. If causal genetic effects exist in these genes, they are too small to be detected by this study.

Tracking the origins of Yakutian horses and the genetic basis for their fast adaptation to subarctic environments.

Yakutia, Sakha Republic, in the Siberian Far East, represents one of the coldest places on Earth, with winter record temperatures dropping below -70 °C. Nevertheless, Yakutian horses survive all year round in the open air due to striking phenotypic adaptations, including compact body conformations, extremely hairy winter coats, and acute seasonal differences in metabolic activities. The evolutionary origins of Yakutian horses and the genetic basis of their adaptations remain, however, contentious. Here, we present the complete genomes of nine present-day Yakutian horses and two ancient specimens dating from the early 19th century and ∼5,200 y ago. By comparing these genomes with the genomes of two Late Pleistocene, 27 domesticated, and three wild Przewalski's horses, we find that contemporary Yakutian horses do not descend from the native horses that populated the region until the mid-Holocene, but were most likely introduced following the migration of the Yakut people a few centuries ago. Thus, they represent one of the fastest cases of adaptation to the extreme temperatures of the Arctic. We find cis-regulatory mutations to have contributed more than nonsynonymous changes to their adaptation, likely due to the comparatively limited standing variation within gene bodies at the time the population was founded. Genes involved in hair development, body size, and metabolic and hormone signaling pathways represent an essential part of the Yakutian horse adaptive genetic toolkit. Finally, we find evidence for convergent evolution with native human populations and woolly mammoths, suggesting that only a few evolutionary strategies are compatible with survival in extremely cold environments.

Evolutionary Genomics and Conservation of the Endangered Przewalski's Horse.

Przewalski's horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.

Pros and cons of methylation-based enrichment methods for ancient DNA.

The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved samples, where both DNA fragmentation and deamination have been limited. This work represents an essential step toward the characterization of ancient methylation signatures, which will help understanding the role of epigenetic changes in past environmental and cultural transitions.

Ancient and modern environmental DNA.

DNA obtained from environmental samples such as sediments, ice or water (environmental DNA, eDNA), represents an important source of information on past and present biodiversity. It has revealed an ancient forest in Greenland, extended by several thousand years the survival dates for mainland woolly mammoth in Alaska, and pushed back the dates for spruce survival in Scandinavian ice-free refugia during the last glaciation. More recently, eDNA was used to uncover the past 50 000 years of vegetation history in the Arctic, revealing massive vegetation turnover at the Pleistocene/Holocene transition, with implications for the extinction of megafauna. Furthermore, eDNA can reflect the biodiversity of extant flora and fauna, both qualitatively and quantitatively, allowing detection of rare species. As such, trace studies of plant and vertebrate DNA in the environment have revolutionized our knowledge of biogeography. However, the approach remains marred by biases related to DNA behaviour in environmental settings, incomplete reference databases and false positive results due to contamination. We provide a review of the field.

Ancient genomics.

The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past.