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Plymouth, MI, United States

Bailey S.E.,Queen Mary, University of London | Mao X.,Queen Mary, University of London | Mao X.,East China Normal University | Struebig M.,Queen Mary, University of London | And 7 more authors.
Biological Journal of the Linnean Society | Year: 2016

Museums hold most of the world's most valuable biological specimens and tissues collected, including type material that is often decades or even centuries old. Unfortunately, traditional museum collection and storage methods were not designed to preserve the nucleic acids held within the material, often reducing its potential viability and value for many genetic applications. High-throughput sequencing technologies and associated applications offer new opportunities for obtaining sequence data from museum samples. In particular, target sequence capture offers a promising approach for recovering large numbers of orthologous loci from relatively small amounts of starting material. In the present study, we test the utility of target sequence capture for obtaining data from museum-held material from a speciose mammalian genus: the horseshoe bats (Rhinolophidae: Chiroptera). We designed a 'bait' for capturing > 3600 genes and applied this to 10 species of horseshoe bat that had been collected between 93 and 7 years ago and preserved using a range of methods. We found that the mean recovery rate per species was approximately 89% of target genes with partial sequence coverage, ranging from 3024 to 3186 genes recovered. On average, we recovered 1206 genes with ≥ 90% sequence coverage, per species. Our findings provide good support for the application of large-scale bait capture across congeneric species spanning approximately 15 Myr of evolution. On the other hand, we observed no clear association between the success of capture and the phylogenetic distance from the bait model, although sample sizes precluded a formal test. © 2016 The Linnean Society of London. Source

Enk J.M.,McMaster University | Devault A.M.,McMaster University | Kuch M.,McMaster University | Murgha Y.E.,MYcroarray | And 3 more authors.
Molecular Biology and Evolution | Year: 2014

We report metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract. Enrichment of the nuclear genome ranged from 1.06-to 18.65-fold, to an apparent maximum threshold of ~80% on-target. This projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design. Source

Delsuc F.,Montpellier University | Gibb G.C.,Montpellier University | Gibb G.C.,Massey University | Kuch M.,McMaster University | And 9 more authors.
Current Biology | Year: 2016

Among the fossils of hitherto unknown mammals that Darwin collected in South America between 1832 and 1833 during the Beagle expedition [1] were examples of the large, heavily armored herbivores later known as glyptodonts. Ever since, glyptodonts have fascinated evolutionary biologists because of their remarkable skeletal adaptations and seemingly isolated phylogenetic position even within their natural group, the cingulate xenarthrans (armadillos and their allies [2]). In possessing a carapace comprised of fused osteoderms, the glyptodonts were clearly related to other cingulates, but their precise phylogenetic position as suggested by morphology remains unresolved [3,4]. To provide a molecular perspective on this issue, we designed sequence-capture baits using in silico reconstructed ancestral sequences and successfully assembled the complete mitochondrial genome of Doedicurus sp., one of the largest glyptodonts. Our phylogenetic reconstructions establish that glyptodonts are in fact deeply nested within the armadillo crown-group, representing a distinct subfamily (Glyptodontinae) within family Chlamyphoridae [5]. Molecular dating suggests that glyptodonts diverged no earlier than around 35 million years ago, in good agreement with their fossil record. Our results highlight the derived nature of the glyptodont morphotype, one aspect of which is a spectacular increase in body size until their extinction at the end of the last ice age. © 2016 Elsevier Ltd. Source

Minty J.J.,University of Michigan | Lesnefsky A.A.,University of Michigan | Lin F.,University of Michigan | Lin F.,Tianjin University | And 11 more authors.
Microbial Cell Factories | Year: 2011

Background: Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress.Results: The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation.Conclusions: We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering. © 2011 Minty et al; licensee BioMed Central Ltd. Source

Murgha Y.,MYcroarray | Beliveau B.,Harvard University | Semrau K.,MYcroarray | Schwartz D.,MYcroarray | And 4 more authors.
BioTechniques | Year: 2015

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection–based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization. © 2015, Eaton Publishing Company. All rights reserved. Source

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