Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans

A computational method for estimating the PCR duplication rate in DNA and RNA-seq experiments.

BackgroundPCR amplification is an important step in the preparation of DNA sequencing libraries prior to high-throughput sequencing. PCR amplification introduces redundant reads in the sequence data and estimating the PCR duplication rate is important to assess the frequency of such reads. Existing computational methods do not distinguish PCR duplicates from "natural" read duplicates that represent independent DNA fragments and therefore, over-estimate the PCR duplication rate for DNA-seq and RNA-seq experiments.ResultsIn this paper, we present a computational method to estimate the average PCR duplication rate of high-throughput sequence datasets that accounts for natural read duplicates by leveraging heterozygous variants in an individual genome. Analysis of simulated data and exome sequence data from the 1000 Genomes project demonstrated that our method can accurately estimate the PCR duplication rate on paired-end as well as single-end read datasets which contain a high proportion of natural read duplicates. Further, analysis of exome datasets prepared using the Nextera library preparation method indicated that 45-50% of read duplicates correspond to natural read duplicates likely due to fragmentation bias. Finally, analysis of RNA-seq datasets from individuals in the 1000 Genomes project demonstrated that 70-95% of read duplicates observed in such datasets correspond to natural duplicates sampled from genes with high expression and identified outlier samples with a 2-fold greater PCR duplication rate than other samples.ConclusionsThe method described here is a useful tool for estimating the PCR duplication rate of high-throughput sequence datasets and for assessing the fraction of read duplicates that correspond to natural read duplicates. An implementation of the method is available at https://github.com/vibansal/PCRduplicates

Correlated patterns of genetic diversity and differentiation across an avian family

Comparative studies of closely related taxa can provide insights into the evolutionary forces that shape genome evolution and the prevalence of convergent molecular evolution. We investigated patterns of genetic diversity and differentiation in stonechats (genus Saxicola), a widely distributed avian species complex with phenotypic variation in plumage, morphology, and migratory behavior, to ask whether similar genomic regions have become differentiated in independent, but closely related, taxa. We used whole-genome pooled sequencing of 262 individuals from 5 taxa and found that levels of genetic diversity and divergence are strongly correlated among different stonechat taxa. We then asked if these patterns remain correlated at deeper evolutionary scales and found that homologous genomic regions have become differentiated in stonechats and the closely related Ficedula flycatchers. Such correlation across a range of evolutionary divergence and among phylogenetically independent comparisons suggests that similar processes may be driving the differentiation of these independently evolving lineages, which in turn may be the result of intrinsic properties of particular genomic regions (e.g., areas of low recombination). Consequently, studies employing genome scans to search for areas important for reproductive isolation or adaptation should account for corresponding regions of differentiation, as these regions may not necessarily represent speciation islands or evidence of local adaptation

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies

The preparation and characterization of three solutions of plasmid DNA containing a ruminant-specific fragment with defined copy number concentrations - Reference Materials: IRMM-AD482a,IRMM-AD482b, IRMM-AD482c

This report describes the processing of a set of plasmid solutions, IRMM-AD482a, b and c. The material was produced following ISO Guide 34:2009. A DNA fragment specific for the identification of ruminant meat were cloned into a pUC18 vector to construct the pIRMM-0103 plasmid. The plasmid was diluted to three different concentration levels. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The materials are intended for the determination of a cut-off value to discriminate positive samples from negative samples by quantitative PCR. As any reference material, the materials can also be used for control charts or validation studies. The RM is available as a set of three plastic tubes containing 1 mL of plasmid solution. The minimum amount of sample to be used is 4 μL.JRC.D.2-Standards for Innovation and sustainable Developmen

Discovering cancer-associated transcripts by RNA sequencing

High-throughput sequencing of poly-adenylated RNA (RNA-Seq) in human cancers shows remarkable potential to identify uncharacterized aspects of tumor biology, including gene fusions with therapeutic significance and disease markers such as long non-coding RNA (lncRNA) species. However, the analysis of RNA-Seq data places unprecedented demands upon computational infrastructures and algorithms, requiring novel bioinformatics approaches. To meet these demands, we present two new open-source software packages - ChimeraScan and AssemblyLine - designed to detect gene fusion events and novel lncRNAs, respectively. RNA-Seq studies utilizing ChimeraScan led to discoveries of new families of recurrent gene fusions in breast cancers and solitary fibrous tumors. Further, ChimeraScan was one of the key components of the repertoire of computational tools utilized in data analysis for MI-ONCOSEQ, a clinical sequencing initiative to identify potentially informative and actionable mutations in cancer patients’ tumors. AssemblyLine, by contrast, reassembles RNA sequencing data into full-length transcripts ab initio. In head-to-head analyses AssemblyLine compared favorably to existing ab initio approaches and unveiled abundant novel lncRNAs, including antisense and intronic lncRNAs disregarded by previous studies. Moreover, we used AssemblyLine to define the prostate cancer transcriptome from a large patient cohort and discovered myriad lncRNAs, including 121 prostate cancer-associated transcripts (PCATs) that could potentially serve as novel disease markers. Functional studies of two PCATs - PCAT-1 and SChLAP1 - revealed cancer-promoting roles for these lncRNAs. PCAT1, a lncRNA expressed from chromosome 8q24, promotes cell proliferation and represses the tumor suppressor BRCA2. SChLAP1, located in a chromosome 2q31 ‘gene desert’, independently predicts poor patient outcomes, including metastasis and cancer-specific mortality. Mechanistically, SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex. Collectively, this work demonstrates the utility of ChimeraScan and AssemblyLine as open-source bioinformatics tools. Our applications of ChimeraScan and AssemblyLine led to the discovery of new classes of recurrent and clinically informative gene fusions, and established a prominent role for lncRNAs in coordinating aggressive prostate cancer, respectively. We expect that the methods and findings described herein will establish a precedent for RNA-Seq-based studies in cancer biology and assist the research community at large in making similar discoveries.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120814/1/mkiyer_1.pd