Martin R. Jones

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

CIViC is a community knowledgebase for expert crowdsourcing the clinical interpretation of variants in cancer

CIViC is an expert-crowdsourced knowledgebase for Clinical Interpretation of Variants in Cancer describing the therapeutic, prognostic, diagnostic and predisposing relevance of inherited and somatic variants of all types. CIViC is committed to open-source code, open-access content, public application programming interfaces (APIs) and provenance of supporting evidence to allow for the transparent creation of current and accurate variant interpretations for use in cancer precision medicine.

RNA-mediated interference as a tool for identifying drug targets.

The nematode Caenorhabditis elegans is the first multicellular organism with a fully sequenced genome. As a model organism, C. elegans is playing a special role in functional genomic analyses because it is experimentally tractable on many levels. Moreover, the lessons learned from C. elegans are often applicable across phyla because many of the key biologic processes involved in development and disease have been well conserved. Many global approaches for analysing gene activity are being pursued in C. elegans. RNA-mediated interference (RNAi) is an efficient high-throughput method to disrupt gene function. The basic technique of RNAi involves introducing sequence-specific double-stranded RNA into C. elegans in order to generate a nonheritable, epigenetic knockout of gene function that phenocopies a null mutation in the targeted gene. This technique drastically reduces the time needed to jump from the identification of an interesting gene sequence to achieving an understanding of its function. Thus, RNAi facilitates the high-throughput functional analysis of gene targets identified during drug discovery. RNAi can also help to identify the biochemical mode of action of a drug or pesticide and to identify other genes encoding products that may respond or interact with specific compounds.

RNA-seq analysis of the C. briggsae transcriptome

Curation of a high-quality gene set is the critical first step in genome research, enabling subsequent analyses such as ortholog assignment, cis-regulatory element finding, and synteny detection. In this project, we have reannotated the genome of Caenorhabditis briggsae, the best studied sister species of the model organism Caenorhabditis elegans. First, we applied a homology-based gene predictor genBlastG to annotate the C. briggsae genome. We then validated and further improved the C. briggsae gene annotation through RNA-seq analysis of the C. briggsae transcriptome, which resulted in the first validated C. briggsae gene set (23,159 genes), among which 7347 genes (33.9% of all genes with introns) have all of their introns confirmed. Most genes (14,812, or 68.3%) have at least one intron validated, compared with only 3.9% in the most recent WormBase release (WS228). Of all introns in the revised gene set (103,083), 61,503 (60.1%) have been confirmed. Additionally, we have identified numerous trans-splicing leaders (SL1 and SL2 variants) in C. briggsae, leading to the first genome-wide annotation of operons in C. briggsae (1105 operons). The majority of the annotated operons (564, or 51.0%) are perfectly conserved in C. elegans, with an additional 345 operons (or 31.2%) somewhat divergent. Additionally, RNA-seq analysis revealed over 10 thousand small-size assembly errors in the current C. briggsae reference genome that can be readily corrected. The revised C. briggsae genome annotation represents a solid platform for comparative genomics analysis and evolutionary studies of Caenorhabditis species.

The North American bullfrog draft genome provides insight into hormonal regulation of long noncoding RNA

Frogs play important ecological roles, and several species are important model organisms for scientific research. The globally distributed Ranidae (true frogs) are the largest frog family, and have substantial evolutionary distance from the model laboratory Xenopus frog species. Unfortunately, there are currently no genomic resources for the former, important group of amphibians. More widely applicable amphibian genomic data is urgently needed as more than two-thirds of known species are currently threatened or are undergoing population declines. We report a 5.8 Gbp (NG50 = 69 kbp) genome assembly of a representative North American bullfrog (Rana [Lithobates] catesbeiana). The genome contains over 22,000 predicted protein-coding genes and 6,223 candidate long noncoding RNAs (lncRNAs). RNA-Seq experiments show thyroid hormone causes widespread transcriptional change among protein-coding and putative lncRNA genes. This initial bullfrog draft genome will serve as a key resource with broad utility including amphibian research, developmental biology, and environmental research.The globally-distributed Ranidae (true frogs) are the largest frog family. Here, Hammond et al. present a draft genome of the North American bullfrog, Rana (Lithobates) catesbeiana, as a foundation for future understanding of true frog genetics as amphibian species face difficult environmental challenges.

The atm-1 gene is required for genome stability in Caenorhabditis elegans

The Ataxia-telangiectasia-mutated (ATM) gene in humans was identified as the basis of a rare autosomal disorder leading to cancer susceptibility and is now well known as an important signal transducer in response to DNA damage. An approach to understanding the conserved functions of this gene is provided by the model system, Caenorhabditis elegans. In this paper we describe the structure and loss of function phenotype of the ortholog atm-1. Using bioinformatic and molecular analysis we show that the atm-1 gene was previously misannotated. We find that the transcript is in fact a product of three gene predictions, Y48G1BL.2 (atm-1), K10E9.1, and F56C11.4 that together make up the complete coding region of ATM-1. We also characterize animals that are mutant for two available knockout alleles, gk186 and tm5027. As expected, atm-1 mutant animals are sensitive to ionizing radiation. In addition, however, atm-1 mutants also display phenotypes associated with genomic instability, including low brood size, reduced viability and sterility. We document several chromosomal fusions arising from atm-1 mutant animals. This is the first time a mutator phenotype has been described for atm-1 in C. elegans. Finally we demonstrate the use of a balancer system to screen for and capture atm-1-derived mutational events. Our study establishes C. elegans as a model for the study of ATM as a mutator potentially leading to the development of screens to identify therapeutic targets in humans.

Successful targeting of the NRG1 pathway indicates novel treatment strategy for metastatic cancer

Background NRG1 fusion-positive lung cancers have emerged as potentially actionable events in lung cancer, but clinical support is currently limited and no evidence of efficacy of this approach in cancers beyond lung has been shown. Patients and methods Here, we describe two patients with advanced cancers refractory to standard therapies. Patient 1 had lung adenocarcinoma and patient 2 cholangiocarcinoma. Whole-genome and transcriptome sequencing were carried out for these cases with select findings validated by fluorescence in situ hybridization. Results Both tumors were found to be positive for NRG1 gene fusions. In patient 1, an SDC4-NRG1 gene fusion was detected, similar gene fusions having been described in lung cancers previously. In patient 2, a novel ATP1B1-NRG1 gene fusion was detected. Cholangiocarcinoma is not a disease type in which NRG1 fusions had been described previously. Integrative genome analysis was used to assess the potential functional significance of the detected genomic events including the gene fusions, prioritizing therapeutic strategies targeting the HER-family of growth factor receptors. Both patients were treated with the pan HER-family kinase inhibitor afatinib and both displayed significant and durable response to treatment. Upon progression sites of disease were sequenced. The lack of obvious genomic events to describe the disease progression indicated that broad transcriptomic or epigenetic mechanisms could be attributed to the lack of prolonged response to afatinib. Conclusion These observations lend further support to the use of pan HER-tyrosine kinase inhibitors for the treatment of NRG1 fusion-positive in both cancers of lung and hepatocellular origin and indicate more broadly that cancers found to be NRG1 fusion-positive may benefit from such a clinical approach regardless of their site of origin. Clinical trial information Personalized Oncogenomics (POG) Program of British Columbia: Utilization of Genomic Analysis to Better Understand Tumour Heterogeneity and Evolution (NCT02155621).

Oligonucleotide Array Comparative Genomic Hybridization (oaCGH) based characterization of genetic deficiencies as an aid to gene mapping in Caenorhabditis elegans

Background:A collection of genetic deficiencies covering over 70% of the Caenorhabditis elegans genome exists, however the application of these valuable biological tools has been limited due to the incomplete correlation between their genetic and physical characterization.Results:We have applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) to the high resolution, molecular characterization of several genetic deficiency and duplication strains in a 5 Mb region of Chromosome III. We incorporate this data into a physical deficiency map which is subsequently used to direct the positional cloning of essential genes within the region. From this analysis we are able to quickly determine the molecular identity of several previously unidentified mutations.Conclusion:We have applied accurate, high resolution molecular analysis to the characterization of genetic mapping tools in Caenorhabditis elegans. Consequently we have generated a valuable physical mapping resource, which we have demonstrated can aid in the rapid molecular identification of mutations of interest.

Response to angiotensin blockade with irbesartan in a patient with metastatic colorectal cancer.

Personalised oncogenomics analysis revealed potential oncogene addiction of the AP-1 transcriptional complex in a chemo-refractory and MMR-deficient tumor from a patient with metastatic colon cancer. Based on this, treatment with the angiotensin receptor agonist irbesartan was initiated to target the renin–angiotensin system upstream of the AP-1 complex, leading to a profound and durable response.

Genomic sequence of a mutant strain of Caenorhabditis elegans with an altered recombination pattern

BackgroundThe original sequencing and annotation of the Caenorhabditis elegans genome along with recent advances in sequencing technology provide an exceptional opportunity for the genomic analysis of wild-type and mutant strains. Using the Illumina Genome Analyzer, we sequenced the entire genome of Rec-1, a strain that alters the distribution of meiotic crossovers without changing the overall frequency. Rec-1 was derived from ethylmethane sulfonate (EMS)-treated strains, one of which had a high level of transposable element mobility. Sequencing of this strain provides an opportunity to examine the consequences on the genome of altering the distribution of meiotic recombination events.ResultsUsing Illumina sequencing and MAQ software, 83% of the base pair sequence reads were aligned to the reference genome available at Wormbase, providing a 21-fold coverage of the genome. Using the software programs MAQ and Slider, we observed 1124 base pair differences between Rec-1 and the reference genome in Wormbase (WS190), and 441 between the mutagenized Rec-1 (BC313) and the wild-type N2 strain (VC2010). The most frequent base-substitution was G:C to A:T, 141 for the entire genome most of which were on chromosomes I or X, 55 and 31 respectively. With this data removed, no obvious pattern in the distribution of the base differences along the chromosomes was apparent. No major chromosomal rearrangements were observed, but additional insertions of transposable elements were detected. There are 11 extra copies of Tc1, and 8 of Tc2 in the Rec-1 genome, most likely the remains of past high-hopper activity in a progenitor strain.ConclusionOur analysis of high-throughput sequencing was able to detect regions of direct repeat sequences, deletions, insertions of transposable elements, and base pair differences. A subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome hybridization. The major phenotype of the Rec-1 strain is an alteration in the preferred position of the meiotic recombination event with no other significant phenotypic consequences. In this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the Rec-1 mutation.