Vrijenhoek T.,University Utrecht |
Kraaijeveld K.,Leiden University |
Elferink M.,University Utrecht |
De Ligt J.,Radboud University Nijmegen |
And 44 more authors.
European Journal of Human Genetics | Year: 2015
Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care. © 2015 Macmillan Publishers Limited All rights reserved.
Gursinsky T.,Martin Luther University of Halle Wittenberg |
Pirovano W.,BaseClear |
Gambino G.,National Research Council Italy |
Friedrich S.,Martin Luther University of Halle Wittenberg |
And 2 more authors.
Plant Physiology | Year: 2015
The plant ARGONAUTE1 protein (AGOl) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGOl -like gene in Nicotiana benthamiana, NbAGOl-lH and NbAGOl-lL. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGOl-l homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGOl mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGOl homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGOl homeolog genes, which has important implications for improving resilience measures of the plant during viral infections. © 2015 American Society of Plant Biologists. All rights reserved.
Lammers Y.,Naturalis Biodiversity Center |
Lammers Y.,Leiden University |
Kremer D.,Leiden University |
Brakefield P.M.,Leiden University |
And 4 more authors.
Molecular Ecology Resources | Year: 2013
Hybrid zones are regions where genetically distinct populations meet, mate and produce offspring. In such zones, genetically less compatible gene combinations are usually generated, resulting in reduced fitness, and hybrid zones are often maintained because of continuous removal of unfit genotypes, balanced by gene flow into the zone from the parental populations (and are then referred to as 'tension zones'). Tension zones often display unexpectedly high frequencies of gene variants that are rare outside the zone. Previous work has shown that this 'rare allele phenomenon' is not the result of intragenic recombination or increased mutation rates. Further understanding of the population genetics of the phenomenon requires an approach in which both the numbers of individuals and the numbers of loci is increased. Here, we report an approach using a combination of Illumina next-generation sequencing and mass spectrophotometer genotyping to identify markers that may be used for genome-wide investigations of the rare allele phenomenon. We test this approach on a hybrid zone in the land snail Albinaria hippolyti from Greece. © 2012 Blackwell Publishing Ltd.
PubMed | VU University Amsterdam and BaseClear
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2016
The increasing importance of Next Generation Sequencing (NGS) techniques has highlighted the key role of multiple sequence alignment (MSA) in comparative structure and function analysis of biological sequences. MSA often leads to fundamental biological insight into sequence-structure-function relationships of nucleotide or protein sequence families. Significant advances have been achieved in this field, and many useful tools have been developed for constructing alignments, although many biological and methodological issues are still open. This chapter first provides some background information and considerations associated with MSA techniques, concentrating on the alignment of protein sequences. Then, a practical overview of currently available methods and a description of their specific advantages and limitations are given, to serve as a helpful guide or starting point for researchers who aim to construct a reliable MSA.