Biology subjects

Glenn, T. C.

Publications and source records attributed to Glenn, T. C..

3 recordsLinked to original sources

bioRxiv2018

Targeted Gene Bisulfite Sequencing Identifies Differential Methylation in p21

Next-generation sequencing (NGS) methods are widely available to assess methylation of whole-genomes, reduced representation of genomes, and target capture of many loci, but simple, flexible, and low-cost methods are needed to leverage NGS for sequencing single-locus amplicons from large numbers of samples. We developed a two-stage PCR approach, targeted gene bisulfite sequencing (TGBS) which uses the Illumina MiSeq and Bismark bisulfite mapper, to assess site specific changes in methylation of the cyclin-dependent kinase inhibitor p21 (CDKN1a) after exposure to a DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (5-Aza) and determine the differences between human and rat p21 methylation. TGBS analysis of human embryonic kidney cells (HEK293) and human proximal tubular cells (hPT) demonstrated variation at a known methylation sensitive site (SIE-1), but not in rat kidney cells. Treatment of cells with 5-Aza altered the methylation of this site in correlation with increased p21 protein expression. We also found that human and rat p21 promoter sequences differ considerably in the amount of basal DNA methylation. These data showed the utility of TGBS for rapid analysis of DNA methylation of specific loci. We provide links to a ready-to-run Virtualbox that includes the program and commands for methylation analysis of bisulfite datasets, including step-by-step directions.

molecular biology↗

bioRxiv2017

Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about $1USD per Sample (3RAD)

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially due to the inability to highly-multiplex samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that relies on adapters and primers designed in Adapterama I. Major features of our method include: 1) minimizing the number of processing steps; 2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; 3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; 4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; 5) interchangeable adapter designs; 6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; 7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, 8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

genomics↗

bioRxiv2017

Conflicting evolutionary histories of the mitochondrial and nuclear genomes in New World Myotis

The rapid diversification of Myotis bats into more than 100 species is one of the most extensive mammalian radiations available for study. Efforts to understand relationships within Myotis have primarily utilized mitochondrial markers and trees inferred from nuclear markers lacked resolution. Our current understanding of relationships within Myotis is therefore biased towards a set of phylogenetic markers that may not reflect the history of the nuclear genome. To resolve this, we sequenced the full mitochondrial genomes of 37 representative Myotis, primarily from the New World, in conjunction with targeted sequencing of 3,648 ultraconserved elements (UCEs). We inferred the phylogeny and explored the effects of concatenation and summary phylogenetic methods, as well as combinations of markers based on informativeness or levels of missing data, on our results. Of the 294 phylogenies generated from the nuclear UCE data, all are significantly different from phylogenies inferred using mitochondrial genomes. Even within the nuclear data, quartet frequencies indicate that around half of all UCE loci conflict with the estimated species tree. Several factors can drive such conflict, including incomplete lineage sorting, introgressive hybridization, or even phylogenetic error. Despite the degree of discordance between nuclear UCE loci and the mitochondrial genome and among UCE loci themselves, the most common nuclear topology is recovered in one quarter of all analyses with strong nodal support. Based on these results, we re-examine the evolutionary history of Myotis to better understand the phenomena driving their unique nuclear, mitochondrial, and biogeographic histories.

evolutionary biology↗