Benchmarking of Amplicon-Based Next-Generation Sequencing Panels Combined with Bioinformatics Solutions for Germline BRCA1 and BRCA2 Alteration Detection

International audienceThe recent deployment of next-generation sequencing approaches in routine laboratory analysis has considerably modified the landscape of BRCA1 and BRCA2 germline alteration detection in patients with a high risk of developing breast and/or ovarian cancer. Several commercial multiplex amplicon-based panels and bioinformatics solutions are currently available. In this study, we evaluated the combinations of several BRCA testing assays and bioinformatics solutions for the identification of single-nucleotide variants, insertion/deletion variants, and copy number variations (CNVs). Four assays (BRCA Tumor, BRCA HC, Ion AmpliSeq BRCA, and Access Array BRCA) and two commercial bioinformatics solutions (SeqNext software version 4.3.1 and Sophia DDM version 5.0.13) were tested on a set of 28 previously genotyped samples. All solutions exhibited accurate detection of single-nucleotide variants and insertion/deletion variants, except for Ion AmpliSeq BRCA, which exhibited a decrease in coverage. Of interest, for CNV analysis, the best accuracy was observed with the Sophia DDM platform regardless of the BRCA kit used. Finally, the performance of the most relevant combination (BRCA Tumor and Sophia DDM) was blindly validated on an independent set of 152 samples. Altogether, our results emphasize the need to accurately compare and control both molecular next-generation sequencing approaches and bioinformatics pipelines to limit the number of discrepant alterations and to provide a powerful tool for reliable detection of genetic alterations in BRCA1 and BRCA2, notably CNVs

Complete genome sequence of Mycobacterium sp. Strain 3519A

International audienceMycobacterium sp. strain 3519A is a nontuberculous mycobacterium isolated from sputum from a Cambodian patient with a pulmonary infection. We report here the first complete 7.3-Mbp-long genome sequence of Mycobacterium sp. 3519A with 66.35% GC content, encoding 7,029 protein-coding genes, 50 tRNAs, and 5 rRNA genes

Complete Genome Sequence of Mycobacterium sp. Strain 4858

International audienceMycobacterium sp. strain 4858 is a nontuberculous mycobacterium isolated from sputum in a Cambodian patient with a pulmonary infection. We report the first complete 5.6-Mbp-long genome sequence of Mycobacterium strain 4858, with 68.24% GC content, carrying 5,255 protein-coding genes, 47 tRNAs, and 3 rRNA genes

Evaluating the Transition from Targeted to Exome Sequencing: A Guide for Clinical Laboratories

The transition from targeted to exome or genome sequencing in clinical contexts requires quality standards, such as targeted sequencing, in order to be fully adopted. However, no clear recommendations or methodology have emerged for evaluating this technological evolution. We developed a structured method based on four run-specific sequencing metrics and seven sample-specific sequencing metrics for evaluating the performance of exome sequencing strategies to replace targeted strategies. The indicators include quality metrics and coverage performance on gene panels and OMIM morbid genes. We applied this general strategy to three different exome kits and compared them with a myopathy-targeted sequencing method. After having achieved 80 million reads, all-tested exome kits generated data suitable for clinical diagnosis. However, significant differences in the coverage and PCR duplicates were observed between the kits. These are two main criteria to consider for the initial implementation with high-quality assurance. This study aims to assist molecular diagnostic laboratories in adopting and evaluating exome sequencing kits in a diagnostic context compared to the strategy used previously. A similar strategy could be used to implement whole-genome sequencing for diagnostic purposes

Société d'histoire du Droit et des Institutions des Pays Flamands, Picards et Wallons. Journées internationales de Luxembourg. 16-18 mai 1985

Pescatore Pierre, Dupont-Bouchat Marie-Sylvie, Petit Roger, Van Peteghem P.P.J.L., Douxchamps-Lefèvre Cécile, Cauchies Jean-Marie, Genicot Léopold, Wijffels A., Lottin Alain, Van den Bergh G. C. J. J., Dams J., Hüdemann C., Sprenger R.M., Van Goethem Herman, Bongert Yvonne, Goedert H., Claeys Ch.E. Société d'histoire du Droit et des Institutions des Pays Flamands, Picards et Wallons. Journées internationales de Luxembourg. 16-18 mai 1985. In: Revue du Nord, tome 68, n°268, Janvier-mars 1986. pp. 225-247

The importance of an integrated genotype-phenotype strategy to unravel the molecular bases of titinopathies

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Long-Reads Sequencing Strategy to Localize Variants in TTN Repeated Domains

International audienceTitin protein is responsible for muscle elasticity. The TTN gene, composed of 364 exons, is subjected to extensive alternative splicing and leads to different isoforms expressed in skeletal and cardiac muscle. Variants in TTN are responsible for myopathies with a wide phenotypic spectrum and autosomal dominant or recessive transmission. The I-band coding domain, highly subject to alternative splicing, contains a three-zone block of repeated sequences with 99% homology. Sequencing and localization of variants in these areas are complex when using short-reads sequencing, a second-generation sequencing technique. We have implemented a protocol based on the third-generation sequencing technology (long-reads sequencing). This new method allows us to localize variants in these repeated areas to improve the diagnosis of TTN-related myopathies and offer the analysis of relatives in postnatal or in prenatal screening

SpliceAI-visual: a free online tool to improve SpliceAI splicing variant interpretation

International audienceAbstract SpliceAI is an open-source deep learning splicing prediction algorithm that has demonstrated in the past few years its high ability to predict splicing defects caused by DNA variations. However, its outputs present several drawbacks: (1) although the numerical values are very convenient for batch filtering, their precise interpretation can be difficult, (2) the outputs are delta scores which can sometimes mask a severe consequence, and (3) complex delins are most often not handled. We present here SpliceAI-visual, a free online tool based on the SpliceAI algorithm, and show how it complements the traditional SpliceAI analysis. First, SpliceAI-visual manipulates raw scores and not delta scores, as the latter can be misleading in certain circumstances. Second, the outcome of SpliceAI-visual is user-friendly thanks to the graphical presentation. Third, SpliceAI-visual is currently one of the only SpliceAI-derived implementations able to annotate complex variants (e.g., complex delins). We report here the benefits of using SpliceAI-visual and demonstrate its relevance in the assessment/modulation of the PVS1 classification criteria. We also show how SpliceAI-visual can elucidate several complex splicing defects taken from the literature but also from unpublished cases. SpliceAI-visual is available as a Google Colab notebook and has also been fully integrated in a free online variant interpretation tool, MobiDetails ( https://mobidetails.iurc.montp.inserm.fr/MD ). Graphical abstrac