Detecting DNA Methylation using the Oxford Nanopore Technologies MinION sequencer, bioRxiv, 2016-04-05
AbstractNanopore sequencing instruments measure the change in electric current caused by DNA transiting through the pore. In experimental and prototype nanopore sequencing devices it has been shown that the electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine. Here we quantify the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. Using synthetically methylated DNA we are able to train a hidden Markov model to distinguish 5-methylcytosine from unmethylated cytosine in DNA. We demonstrate by sequencing natural human DNA, without any special library preparation, that global patterns of methylation can be detected from low-coverage sequencing and that the methylation status of CpG islands can be reliably predicted from single MinION reads. Our trained model and prediction software is open source and freely available to the community under the MIT license.
biorxiv genomics 200-500-users 2016Real time selective sequencing using nanopore technology., bioRxiv, 2016-02-04
The Oxford Nanopore MinION is a portable real time sequencing device which functions by sensing the change in current flow through a nanopore as DNA passes through it. These current values can be streamed in real time from individual nanopores as DNA molecules traverse them. Furthermore, the technology enables individual DNA molecules to be rejected on demand by reversing the voltage across specific channels. In theory, combining these features enables selection of individual DNA molecules for sequencing from a pool, an approach called Read Until. Here we apply dynamic time warping to match short query current traces to references, demonstrating selection of specific regions of small genomes, individual amplicons from a group of targets, or normalisation of amplicons in a set. This is the first demonstration of direct selection of specific DNA molecules in real time whilst sequencing on any device and enables many novel uses for the MinION.
biorxiv genomics 200-500-users 2016No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini, bioRxiv, 2015-12-02
AbstractTardigrades are meiofaunal ecdysozoans that are key to understanding the origins of Arthropoda. Many species of Tardigrada can survive extreme conditions through cryptobiosis. In a recent paper (Boothby TC et al (2015) Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade. Proc Natl Acad Sci USA 11215976-15981) the authors concluded that the tardigrade Hypsibius dujardini had an unprecedented proportion (17%) of genes originating through functional horizontal gene transfer (fHGT), and speculated that fHGT was likely formative in the evolution of cryptobiosis. We independently sequenced the genome of H. dujardini. As expected from whole-organism DNA sampling, our raw data contained reads from non-target genomes. Filtering using metagenomics approaches generated a draft H. dujardini genome assembly of 135 Mb with superior assembly metrics to the previously published assembly. Additional microbial contamination likely remains. We found no support for extensive fHGT. Among 23,021 gene predictions we identified 0.2% strong candidates for fHGT from bacteria, and 0.2% strong candidates for fHGT from non-metazoan eukaryotes. Cross-comparison of assemblies showed that the overwhelming majority of HGT candidates in the Boothby et al. genome derived from contaminants. We conclude that fHGT into H. dujardini accounts for at most 1-2% of genes and that the proposal that one sixth of tardigrade genes originate from functional HGT events is an artefact of undetected contamination.
biorxiv genomics 200-500-users 2015Analysis of protein-coding genetic variation in 60,706 humans, bioRxiv, 2015-10-31
SummaryLarge-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) sequence data for 60,706 individuals of diverse ethnicities generated as part of the Exome Aggregation Consortium (ExAC). The resulting catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We show that this catalogue can be used to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; we identify 3,230 genes with near-complete depletion of truncating variants, 72% of which have no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human “knockout” variants in protein-coding genes.
biorxiv genomics 200-500-users 2015Relative Citation Ratio (RCR) A new metric that uses citation rates to measure influence at the article level, bioRxiv, 2015-10-23
AbstractDespite their recognized limitations, bibliometric assessments of scientific productivity have been widely adopted. We describe here an improved method that makes novel use of the co-citation network of each article to field-normalize the number of citations it has received. The resulting Relative Citation Ratio is article-level and field-independent, and provides an alternative to the invalid practice of using Journal Impact Factors to identify influential papers. To illustrate one application of our method, we analyzed 88,835 articles published between 2003 and 2010, and found that the National Institutes of Health awardees who authored those papers occupy relatively stable positions of influence across all disciplines. We demonstrate that the values generated by this method strongly correlate with the opinions of subject matter experts in biomedical research, and suggest that the same approach should be generally applicable to articles published in all areas of science. A beta version of iCite, our web tool for calculating Relative Citation Ratios of articles listed in PubMed, is available at <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsicite.od.nih.gov>httpsicite.od.nih.gov<jatsext-link>.
biorxiv scientific-communication-and-education 200-500-users 2015TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants, bioRxiv, 2015-10-07
SUMMARYA major constraint on the evolution of large body sizes in animals is an increased risk of developing cancer. There is no correlation, however, between body size and cancer risk. This lack of correlation is often referred to as ‘Peto’s Paradox’. Here we show that the elephant genome encodes 20 copies of the tumor suppressor gene TP53 and that the increase in TP53 copy number occurred coincident with the evolution of large body sizes, the evolution of extreme sensitivity to genotoxic stress, and a hyperactive TP53 signaling pathway in the elephant (Proboscidean) lineage. Furthermore we show that several of the TP53 retrogenes (TP53RTGs) are transcribed and likely translated. While TP53RTGs do not appear to directly function as transcription factors, they do contribute to the enhanced sensitivity of elephant cells to DNA damage and the induction of apoptosis by regulating activity of the TP53 signaling pathway. These results suggest that an increase in the copy number of TP53 may have played a direct role in the evolution of very large body sizes and the resolution of Peto’s paradox in Proboscideans.
biorxiv genetics 200-500-users 2015