Suchergebnisse

Population genomics is a fast-developing discipline with promising applications in a growing number of life sciences fields. Advances in sequencing technologies and bioinformatics tools allow population genomics to exploit genome-wide information to identify the molecular variants underlying traits of interest and the evolutionary forces that modulate these variants through space and time. However, the cost of genomic analyses of multiple populations is still too high to address them through individual genome sequencing. Pooling individuals for sequencing can be a more effective strategy in Single Nucleotide Polymorphism (SNP) detection and allele frequency estimation because of a higher total coverage. However, compared to individual sequencing, SNP calling from pools has the additional difficulty of distinguishing rare variants from sequencing errors, which is often avoided by establishing a minimum threshold allele frequency for the analysis. Finding an optimal balance between minimizing information loss and reducing sequencing costs is essential to ensure the success of population genomics studies. Here, we have benchmarked the performance of SNP callers for Pool-seq data, based on different approaches, under different conditions, and using computer simulations and real data. We found that SNP callers performance varied for allele frequencies up to 0.35. We also found that SNP callers based on Bayesian (SNAPE-pooled) or maximum likelihood (MAPGD) approaches outperform the two heuristic callers tested (VarScan and PoolSNP), in terms of the balance between sensitivity and FDR both in simulated and sequencing data. Our results will help inform the selection of the most appropriate SNP caller not only for large-scale population studies but also in cases where the Pool-seq strategy is the only option, such as in metagenomic or polyploid studies. ; This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (H2020-ERC-2014-CoG-647900). ; Peer reviewed

In this chapter we will review the common steps in the analysis of whole genome singlebase-pair resolution methylation data including the pre-processing of the reads, the alignment and the read out of the methylation information of individual cytosines. We will specially focus on the possible error sources which need to be taken into account in order to generate high quality methylation maps. Several tools have been already developed to convert the sequencing data into knowledge about the methylation levels. We will review the most used tools discussing both technical aspects like user-friendliness and speed, but also biologically relevant questions as the quality control. For one of these tools, NGSmethPipe, we will give a step by step tutorial including installation and methylation profiling for different data types and species. We will conclude the chapter with a brief discussion of NGSmethDB, a database for the storage of single-base resolution methylation maps that can be used to further analyze the obtained methylation maps. ; This work was supported by the Ministry of Innovation and Science of the Spanish Government [BIO2010-20219 (M.H.), BIO2008-01353 (J.L.O.)]; 'Juan de la Cierva' grant (to M.H.) and Basque Country 'Programa de formación de investigadores' grant (to G.B.).

Abstract High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.

High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security. ; Peer reviewed

Abstract High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security. ; Peer Reviewed

Twin studies have long provided a means to separate the contributions of genetic and environmental factors. A recent pioneering report by Baranzini et al. presented an analysis of the complete genomes and epigenomes of a monozygotic (MZ) twin pair discordant for multiple sclerosis. This failed to find any difference between the twins, raising doubts regarding the value of whole-genome twin studies for defining disease susceptibility alleles. However, the study was carried out with DNA extracted from blood. In many cases, the hematopoietic lineages of MZ twins are chimeric due to twin-to-twin exchange of hematopoietic stem cells during embryogenesis. We therefore wondered how chimerism might impact the ability to identify genetic differences. We inferred the blood chimerism rates and profiles of more than 30 discordant twin cases from a wide variety of medical conditions. We found that the genotype compositions of the twins were highly similar. We then benchmarked the performance of SNP callers to detect discordant variations using high-throughput sequencing data. Our analysis revealed that chimerism patterns, well within the range normally observed in MZ twins, greatly reduce the sensitivity of SNP calls. This raises questions regarding any conclusions of genomic homogeneity that might be drawn from studies of blood-derived twin DNA.

WOS: 000366702100002 ; PubMed ID: 26684649 ; Background: Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics. Methods: A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes. Results: Analyses revealed a mutation detection sensitivity of 97.3 %, an average coverage per gene of 98.0 %, and adequate coverage over 98.6 % of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38 %). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals. Conclusions: We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization. ; European Research Council under the European Union's Seventh Framework Programme (FP)/ERC [311335]; Swedish Research Council; Swedish Children's Cancer Foundation; Swedish Cancer Foundation; Swedish Foundation for Strategic Research; Wallenberg Foundation; Stockholm County Council (ALF project); Board of Postgraduate Studies at Karolinska Institutet ; The authors thank the physicians that referred the patients for genetic and immunological evaluations and Nina Jantti and Anh Nhi Tran for technical assistance. This study was supported by the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement no. 311335, Swedish Research Council, Swedish Children's Cancer Foundation, Swedish Cancer Foundation, Swedish Foundation for Strategic Research, Wallenberg Foundation and the Stockholm County Council (ALF project). B.T. was supported by a PhD student scholarship awarded by the Board of Postgraduate Studies at Karolinska Institutet.

ZusammenfassungTrinkwassergewinnung mittels Uferfiltration gewinnt weltweit und auch in Österreich zunehmend an Bedeutung. Entlang der Donau existieren verschiedene Uferfiltratstandorte, deren Brunnen zu verschiedenen saisonalen Zeitpunkten 2016 und 2017 mittels High-Throughput-Sequencing (Ermittlung der mikrobiellen Zusammensetzung bzw. des Mikrobioms) und mittels Durchflusszytometrie (Gesamtzellzahl) untersucht wurden. Dabei konnten räumliche Unterschiede zwischen den Brunnenfeldern, saisonale Änderungen sowie insbesondere Einflüsse durch Hochwasserereignisse festgestellt werden. Die Erweiterung der Datenbasis durch die Hinzunahme von Sequenzierungsdaten einer Donau-Studie zeigt interessante Möglichkeiten, vorhandenes Wissen mit bioinformatischen Mitteln einzubinden und neue Erkenntnisse zu generieren.

ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO–sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs. To better understand the biological functions of AGO–sRNA complexes, it is crucial to identify the repertoire of target RNAs they regulate. Here I present a detailed AGO–RNA IP followed by high-throughput sequencing (AGO RIP-Seq) methodology for the isolation of AGO ternary complexes from plant tissues and the high-throughput sequencing of AGO-bound target RNAs. In particular, the protocol describes the IP of slicer-deficient hemagglutinin (HA)-tagged AGO proteins expressed in plant tissues, the isolation of AGO-bound RNAs, and the generation of target RNA libraries for high-throughput sequencing. ; This work was supported by an Individual Fellowship from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 655841 to A.C. ; Carbonell Olivares, A. (2017). Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants. En Plant Argonaute Proteins: Methods and Protocols. Springer Link. 93-112. https://doi.org/10.1007/978-1-4939-7165-7_6 ; 93 ; 112 ; Carbonell A, Carrington JC (2015) Antiviral roles of plant ARGONAUTES. Curr Opin Plant Biol 27:111–117. doi:10.1016/j.pbi.2015.06.013 ; Fang X, Qi Y (2016) RNAi in plants: an Argonaute-centered view. Plant Cell 28(2):272–285. doi:10.1105/tpc.15.00920 ; Vaucheret H (2008) Plant ARGONAUTES. Trends Plant Sci 13(7):350–358. doi:10.1016/j.tplants.2008.04.007 ...

High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security. ; Published version

1: Introduction -- 2: Basic Structure -- 2.1 Problem Definition -- 2.2 Problem Formulation -- 2.3 Problem Assumptions -- 3: Measures of Performance -- 3.1 Characteristics of Criteria -- 3.2 Single Criteria -- 3.3 Multiple Criteria -- 4: Combinatorial Aspects -- 4.1 Characteristics of Sequences -- 4.2 Characteristics of Schedules -- 5: Statistical Aspects -- 5.1 Theoretical Study I -- 5.2 Theoretical Study II.