Detection of Genomic Structural Variants from Next-Generation Sequencing Data

Structural variants are genomic rearrangements larger than 50?bp accounting for around 1% of the variation among human genomes. They impact on phenotypic diversity and play a role in various diseases including neurological/neurocognitive disorders and cancer development and progression. Dissecting structural variants from next-generation sequencing data presents several challenges and a number of approaches have been proposed in the literature. In this mini review, we describe and summarize the latest tools ? and their underlying algorithms ? designed for the analysis of whole-genome sequencing, whole-exome sequencing, custom captures, and amplicon sequencing data, pointing out the major advantages/drawbacks. We also report a summary of the most recent applications of third-generation sequencing platforms. This assessment provides a guided indication ? with particular emphasis on human genetics and copy number variants ? for researchers involved in the investigation of these genomic events

Diagnostic applications of next generation sequencing: working towards quality standards

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workflows and discuss possible approaches to define quality criteria for NGS in molecular genetic diagnostics

Microarrays in molecular profiling of cancer : focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.Biolääketieteessä mikrosiruilla tutkitaan samanaikaisesti tuhansia molekyylejä solu- tai kudosnäytteestä. Mikrosirut koostuvat kiinteällä alustalla, kuten mikroskooppilasilla, olevista tuhansista pienistä pisteistä. Jokainen piste voi sisältää esimerkiksi 25-60 emäksen pituisia oligonukleotidejä, jotka vastaavat tiettyä geeniä. Näin mikrosirujen avulla voidaan tutkia vaikkapa useiden geenien ilmentymistä näytteestä. Mikrosiruilla on paljon sovelluksia biolääketieteen alalla. Erityisesti siruja on käytetty syöpätutkimuksessa. Mikrosiruja geenien ilmentymisen määrittämiseen valmistetaan paikallisesti tutkimuslaboratoriossa tai ostetaan kaupallisilta valmistajilta. Kaupallisia valmistajia on useita. Monimuotoisuus asettaa haasteita tiedon keräämiselle eri sirutyypeiltä ja kerätyn tiedon vertaamiselle. Tässä väitöskirjatyössä verrattiin geenien ilmentymisen tuloksia kolmelta erityyppiseltä mikrosirulta. Joukossa oli kaksi kaupallista sekä yksi itsetehty siru. Vertailu osoitti, että kaupalliset sirut antoivat samankaltaisempia tuloksia, korrelaatio sirujen välillä 0.78-0.86, kuin itsetehty siru. Vaikka tulokset osoittivat, että eri siruilta voidaan saada vertailukelpoista tietoa, ei vertailu tulosten välillä ole suoraviivaista. Haasteena ovat mikrosirujen tekniset eroavaisuudet sekä tiedon saattaminen vertailukelpoiseen muotoon. Mikrosirujen kehittyessä tutkimustyökalusta kliinisiin sovelluksiin standardoinnilla - ja sitä kautta tulosten paremmalla vertautuvuudella - on tärkeä tehtävä. Työssä tutkittiin myös biologisen tiedon yhdistämistä eri mikrosirumenetelmistä pään ja kaulan alueen syövässä. Mittaamalla mikrosiruilla geenin kopiomäärän muutoksia DNA-tasolla ja yhdistämällä tämä tieto geenin ilmentymistason muutoksiin saatiin tietoa syövälle merkityksellisistä geeneistä. Erityisesti korkeasteiset kopioluvun muutokset vaikuttivat geenien ilmentymiseen, sillä 14-32% näillä alueilla sijaisevista geeneistä oli myös kohonnut ilmentymistaso. Tilastollisin menetelmin osoitettiin satoja geenejä, joilla kopioluvun ja ilmentymän muutos näyttävät olevan yhteydessä toisiinsa. Tässä joukossa oli uusia kohdegeenejä ennestään tunnetuille kromosomialueille, kuten FADD ja PPFIA1 geenit kromosomissa 11. Työssä tunnistetut geenit antavat hyvän pohjan jatkotutkimuksille, jotka voivat vuosien kuluessa johtaa edistysaskeliin pään ja kaulan alueen syövän hoidossa

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia. The Cancer Genome Atlas Research Network has demonstrated the increasing genomic complexity of acute myeloid leukemia (AML). In addition, the network has facilitated our understanding of the molecular events leading to this deadly form of malignancy for which the prognosis has not improved over past decades. AML is a highly heterogeneous disease, and cytogenetics and molecular analysis of the various chromosome aberrations including deletions, duplications, aneuploidy, balanced reciprocal translocations and fusion of transcription factor genes and tyrosine kinases has led to better understanding and identification of subgroups of AML with different prognoses. Furthermore, molecular classification based on mRNA expression profiling has facilitated identification of novel subclasses and defined high-, poor-risk AML based on specific molecular signatures. However, despite increased understanding of AML genetics, the outcome for AML patients whose number is likely to rise as the population ages, has not changed significantly. Until it does, further investigation of the genomic complexity of the disease and advances in drug development are needed. In this review, leading AML clinicians and research investigators provide an up-to-date understanding of the molecular biology of the disease addressing advances in diagnosis, classification, prognostication and therapeutic strategies that may have significant promise and impact on overall patient survival

The AURORA pilot study for molecular screening of patients with advanced breast cancer–a study of the breast international group

Several studies have demonstrated the feasibility of molecular screening of tumour samples for matching patients with cancer to targeted therapies. However, most of them have been carried out at institutional or national level. Herein, we report on the pilot phase of AURORA (NCT02102165), a European multinational collaborative molecular screening initiative for advanced breast cancer patients. Forty-one patients were prospectively enroled at four participating centres across Europe. Metastatic tumours were biopsied and profiled using an Ion Torrent sequencing platform at a central facility. Sequencing results were obtained for 63% of the patients in real-time with variable turnaround time stemming from delays between patient consent and biopsy. At least one clinically actionable mutation was identified in 73% of patients. We used the Illumina sequencing technology for orthogonal validation and achieved an average of 66% concordance of substitution calls per patient. Additionally, copy number aberrations inferred from the Ion Torrent sequencing were compared to single nucleotide polymorphism arrays and found to be 59% concordant on average. Although this study demonstrates that powerful next generation genomic techniques are logistically ready for international molecular screening programs in routine clinical settings, technical challenges remain to be addressed in order to ensure the accuracy and clinical utility of the genomic data.info:eu-repo/semantics/publishe

Detection of deleterious on-target effects after CRISPR-mediated genome editing in human induced pluripotent stem cells

The CRISPR/Cas9 system is an exceedingly powerful technology for precise genome editing. Its ease of use, high editing efficiency and an ever-growing CRISPR-based toolbox has provided researchers with novel possibilities to unravel the molecular and systemic consequences of changes in the genetic code. For this reason, CRISPR is now applied for editing in a wide range of different cell lines and organisms for basic and translational research. Here, accurate and precise editing is an indispensable prerequisite to generate reliable research models. However, a lot remains to be understood about the molecular mechanism of double-strand-breaks (DSBs) in the DNA as introduced by the Cas9 nuclease during editing. In fact, CRISPR editing can be accompanied by inadvertent genomic changes at the targeted locus (on-target) as well as other genomic sites (off-target). These can have drastic consequences on gene activity or expression and therefore need to be carefully investigated. Characterizing and avoiding unwanted off-target effects (OffTE) has been the focus of several studies and reliable tools for their detection have been developed. This is, however, not the case for on-target effects (OnTE) that have only been reported very recently. These can be large deletions, large insertions, complex rearrangements, or regions of copy-neutral loss of heterozygosity (LOH) around the target site. Several studies have described frequent occurrence of OnTEs in mice, but it has not been investigated if clinically relevant human cells, such as induced pluripotent stem cells (iPSCs) are also affected. The main problem with OnTEs is that they often remain unnoticed in standard quality controls like Sanger genotyping of the target locus, and additional checks are lacking in most CRISPR-based studies. This is also because there are no simple detection tools available. Therefore, in this study, we developed simple and reliable tools for OnTE detection after CRISPR genome editing: Structural alterations like large deletions, large insertions or complex rearrangements can be identified by quantifying the number of intact alleles at the edited locus using our new method called quantitative genotyping PCR (qgPCR). In addition, we validated genotyping of neighboring single nucleotide polymorphisms (SNPs) either by Sanger sequencing or SNP microarrays to reveal editing-induced regions of LOH. The entire workflow is broadly applicable to different cell lines and organisms after editing by the NHEJ or HDR pathway. We have applied our newly established detection technology to human iPSCs after HDR-mediated editing and demonstrate universal occurrence of OnTEs at multiple loci in up to 40% of edited single-cell clones. Furthermore, using an in vitro model of Alzheimer’s disease, we illustrate deleterious consequences of OnTEs on expression of the edited gene that may reduce pathogenic effects and therefore interfere with experimental findings. Overall, the threat of undetected OnTEs undermining the reliability of CRISPR-based studies has not received sufficient attention in the field so far. With this thesis, we hope to raise further awareness and propose that our simple and reliable on-target quality control workflow should be an essential part of all relevant genome editing experiments

A multi-modal data resource for investigating topographic heterogeneity in patient-derived xenograft tumors.

Patient-derived xenografts (PDXs) are an essential pre-clinical resource for investigating tumor biology. However, cellular heterogeneity within and across PDX tumors can strongly impact the interpretation of PDX studies. Here, we generated a multi-modal, large-scale dataset to investigate PDX heterogeneity in metastatic colorectal cancer (CRC) across tumor models, spatial scales and genomic, transcriptomic, proteomic and imaging assay modalities. To showcase this dataset, we present analysis to assess sources of PDX variation, including anatomical orientation within the implanted tumor, mouse contribution, and differences between replicate PDX tumors. A unique aspect of our dataset is deep characterization of intra-tumor heterogeneity via immunofluorescence imaging, which enables investigation of variation across multiple spatial scales, from subcellular to whole tumor levels. Our study provides a benchmark data resource to investigate PDX models of metastatic CRC and serves as a template for future, quantitative investigations of spatial heterogeneity within and across PDX tumor models

Computational Biology-Driven Genomic and Epigenomic Delineation of Acute Myeloid Leukemia

Hematopoiesis is the deterministic process of blood cell formation taking place in the bone marrow. Mature blood cells are produced by a tightly controlled mechanism from hematopoietic stem cells (HSCs) residing in the bone marrow. Upon maturation blood cells are released into the peripheral blood and from this point onward can be transported to the different locations of the body. The mature blood cells exert different functions dependent on a strictly controlled path of maturation. The distinct leukocytes comprising granulocytes, monocytes, macrophages, natural killer cells and lymphocytes are essential for the defense against pathogens and foreign invaders, erythrocytes play a pivotal role in the transportation of oxygen to remote organs, and platelets confer the process of blood clotting. Mature blood cells are short-lived and require continuous replenishment. The control of the production and the total number of blood cells is conferred by multipotent progenitors and a small population of pluripotent HSCs (Figure 1). HSCs reside in the bone marrow of adult mammals at the apex of a hierarchy of progenitors which become progressively restricted to several and eventually single lineages of blood cells. Additionally these pluripotent stem cells have the unique ability to self-renew, generating a source for continuous replenishment of the complete blood cell system. The hematopoietic stem cell compartment contains stem cells with progressively decreased self-renewal capacity with the retention of multi-lineage reconstitution. The rare long term HSC (LT-HSC) is at the pinnacle of the hematopoietic hierarchy and is mainly quiescent. With the most conserved rate of self-renewal it prevents the depletion of the stem cell pool. The less rare short term HSC (ST-HSC) still retains a minimal ability for self-renewal and is the more active effector cell for hematopoietic replenishment in normal situations. The main constituent of the hematopoietic stem cell compartment is the multipotent progenitor (MPP) which lost its self-renewal capacity, however, kept the ability to give rise to daughter cells of different lineages. The daughter cells, common myeloid progenitor (CMP) and common lymphoid progenitor (CLP), are still oligopotent as they give rise to multiple blood cell types, e.g., lymphocytes, granulocytes, platelets and erythrocytes. The production of mature blood cells is a strictly controlled process that adapts to the needs of human physiology, e.g., erythrocyte production after blood loss. The control is asserted mainly by external stimuli, e.g., hematopoietic cytokines or growth factors, which are produced by constituents of the regulatory microenvironment within the bone marrow niche, other blood cells or cytokine secreting organs. The microenvironment plays a pivotal role in the formation of adequate numbers of blood cells of the correct type and the hematopoietic cytokines it produces allows the hematopoietic system to dynamically adapt to extramedullary events, e.g., blood loss, infection or cancer immunoediting