Group sequencing around a common due date

Author name used in this publication: C. T. Ng2007-2008 > Academic research: refereed > Publication in refereed journalAccepted ManuscriptPublishe

MinION Analysis and Reference Consortium: Phase 1 data release and analysis

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance

Rapid detection of BRCA1/2 recurrent mutations in Chinese breast and ovarian cancer patients with multiplex SNaPshot genotyping panels.

BRCA1/2 mutations are significant risk factors for hereditary breast and ovarian cancer (HBOC), its mutation frequency in HBOC of Chinese ethnicity is around 9%, in which nearly half are recurrent mutations. In Hong Kong and China, genetic testing and counseling are not as common as in the West. To reduce the barrier of testing, a multiplex SNaPshot genotyping panel that targeted 25 Chinese BRCA1/2 mutation hotspots was developed, and its feasibility was evaluated in a local cohort of 441 breast and 155 ovarian cancer patients. For those who tested negative, they were then subjected to full-gene testing with next-generation sequencing (NGS). BRCA mutation prevalence in this cohort was 8.05% and the yield of the recurrent panel was 3.52%, identifying over 40% of the mutation carriers. Moreover, from 79 Chinese breast cancer cases recruited overseas, 2 recurrent mutations and one novel BRCA2 mutation were detected by the panel and NGS respectively. The developed genotyping panel showed to be an easy-to-perform and more affordable testing tool that can provide important contributions to improve the healthcare of Chinese women with cancer as well as family members that harbor high risk mutations for HBOC

Independent large scale duplications in multiple M. tuberculosis lineages overlapping the same genomic region

Mycobacterium tuberculosis, the causative agent of most human tuberculosis, infects one third of the world's population and kills an estimated 1.7 million people a year. With the world-wide emergence of drug resistance, and the finding of more functional genetic diversity than previously expected, there is a renewed interest in understanding the forces driving genome evolution of this important pathogen. Genetic diversity in M. tuberculosis is dominated by single nucleotide polymorphisms and small scale gene deletion, with little or no evidence for large scale genome rearrangements seen in other bacteria. Recently, a single report described a large scale genome duplication that was suggested to be specific to the Beijing lineage. We report here multiple independent large-scale duplications of the same genomic region of M. tuberculosis detected through whole-genome sequencing. The duplications occur in strains belonging to both M. tuberculosis lineage 2 and 4, and are thus not limited to Beijing strains. The duplications occur in both drug-resistant and drug susceptible strains. The duplicated regions also have substantially different boundaries in different strains, indicating different originating duplication events. We further identify a smaller segmental duplication of a different genomic region of a lab strain of H37Rv. The presence of multiple independent duplications of the same genomic region suggests either instability in this region, a selective advantage conferred by the duplication, or both. The identified duplications suggest that large-scale gene duplication may be more common in M. tuberculosis than previously considere

A nonhuman primate model of inherited retinal disease.

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general

Shotgun Mitogenomics Provides a Reference Phylogenetic Framework and Timescale for Living Xenarthrans

Xenarthra (armadillos, sloths, and anteaters) constitutes one of the four major clades of placental mammals. Despite their phylogenetic distinctiveness in mammals, a reference phylogeny is still lacking for the 31 described species. Here we used Illumina shotgun sequencing to assemble 33 new complete mitochondrial genomes, establishing Xenarthra as the first major placental clade to be fully sequenced at the species level for mitogenomes. The resulting data set allowed the reconstruction of a robust phylogenetic framework and timescale that are consistent with previous studies conducted at the genus level using nuclear genes. Incorporating the full species diversity of extant xenarthrans points to a number of inconsistencies in xenarthran systematics and species definition. We propose to split armadillos in two distinct families Dasypodidae (dasypodines) and Chlamyphoridae (euphractines, chlamyphorines, and tolypeutines) to better reflect their ancient divergence, estimated around 42 million years ago. Species delimitation within long-nosed armadillos (genus Dasypus) appeared more complex than anticipated, with the discovery of a divergent lineage in French Guiana. Diversification analyses showed Xenarthra to be an ancient clade with a constant diversification rate through time with a species turnover driven by high but constant extinction. We also detected a significant negative correlation between speciation rate and past temperature fluctuations with an increase in speciation rate corresponding to the general cooling observed during the last 15 million years. Biogeographic reconstructions identified the tropical rainforest biome of Amazonia and the Guianan shield as the cradle of xenarthran evolutionary history with subsequent dispersions into more open and dry habitats.Fil: Gibb, Gillian C.. Universite de Montpellier; Francia. Massey Universit; Nueva ZelandaFil: Condamine, Fabien L.. University of Gothenburg; Suecia. Universite de Montpellier; Francia. University of Alberta; CanadáFil: Kuch, Melanie. McMaster University; CanadáFil: Enk, Jacob. McMaster University; CanadáFil: Moraes Barros, Nadia. Universidade Do Porto; Portugal. Universidade de Sao Paulo; BrasilFil: Superina, Mariella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Poinar, Hendrik N.. McMaster University; CanadáFil: Delsuc, Frederic. Universite de Montpellier; Franci